Human and non-human animal use of microbial anaplerotic oil

ABSTRACT

Disclosed are techniques and systems for producing microbials having anaplerotic oils that are rich in odd-chain fatty acids, and other beneficial components, at higher concentrations than those present in other natural dietary sources of OCFA, at lower cost, and higher production yield. Further, disclosed are examples of incorporation of these higher concentration OCFA products into food for human and non-human animal consumption.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/240,667, filed Apr. 26, 2021, which is a continuation of U.S. patentapplication Ser. No. 17/090,589 (now U.S. Pat. No. 10,986,856, issuedApr. 27, 2021), filed Nov. 5, 2020, which is a continuation of U.S.patent application Ser. No. 16/720,972 (now U.S. Pat. No. 10,874,131,issued Dec. 29, 2020), filed Dec. 19, 2019, which claims priority toU.S. Provisional Patent Application No. 62/783,546, filed Dec. 21, 2018,and is a continuation-in-part of U.S. patent application Ser. No.16/229,551 (now U.S. Pat. No. 10,745,724, issued Aug. 18, 2020), filedDec. 21, 2018, which claims priority to U.S. Provisional PatentApplication No. 62/731,476, filed Sep. 14, 2018, and U.S. ProvisionalPatent Application No. 62/609,700, filed Dec. 22, 2017.

FIELD OF THE INVENTION

The present invention generally relates to uses for microalgae oil richin odd-chain fatty acid, and more specifically, to human and non-humananimal uses of microbial anaplerotic oil.

BACKGROUND

The citric acid cycle can govern the energy metabolism in aerobicorganisms. In addition, the cycle can provide precursors forbiosynthesis of several amino acids, lipids, chlorophyll and othergrowth-related metabolites. The citric acid cycle is non-catalytic,which means that molecules used in biosynthesis need to be replenishedso that the cycle can keep generating energy. Regardless of how muchacetyl CoA is fed into the citric acid cycle, the cycle is able toproduce merely a limited amount of citric acid intermediates.Anaplerotic substrates can be used to produce intermediates that areused to replenish the oxidative capacity of the citric acid cycle.

Anaplerosis refers to the process of replenishing the citric acid cycleintermediates and restoring energy balance of the cell (metabolichomeostasis). Odd-chain fatty acids (OCFA) can be considered anapleroticbecause, along with acetate units, they can also release propionic acidwhich can enter the citric acid cycle through the methylmalonate pathway(OCFA catabolism). Typical dietary sources of OCFA are milk and butter,but they have only trace amounts (<2% total fatty acids, TFA) ofpentadecanoic (C15:0) and heptadecanoic (C17:0) acid. Syntheticallyproduced concentrated sources, such as tripentanoin and triheptanoin(e.g., oils containing C5:0 and C7:0), are not considered nutritionallipids. Further, current methods that involve the use of Yarrowialipolytica to produce odd chain fatty acids utilize geneticmodification. Specifically, for example, these methods utilize thedeletion of the PHD1 gene in order to improve lipid accumulation. Ref10.

SUMMARY

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key factors oressential features of the claimed subject matter, nor is it intended tobe used to limit the scope of the claimed subject matter.

Disclosed are compositions rich in odd-chain fatty acids (OCFA),including pentadecanoic and heptadecanoic fatty acids, and products richin tridecanoic, pentadecanoic and heptadecanoic fatty acids derived frommicroalgae, yeast or fungi; OCFA promoters that can be used to induceOCFA production; and processes that help reduce an amount of propionateused in OCFA production. Further, techniques and systems can utilizeOCFA rich anaplerotic oils from cultured microbials as supplements oradditives to foods as a nutritional supplement. For example, suchsupplements may be used to replace significant amounts of the dietaryeven chain saturated fat with odd chain saturate fat, withoutsignificantly changing the dietary habits of the consumer. In thisexample, the supplements described herein can be incorporated both as adirect food ingredient and in many different food products. Further,techniques and systems can be used to identify the benefits ofconsumption of these compositions rich in OCFA.

Further, techniques and systems are disclosed for identifying propionicacid toxicity in some types of microorganisms, for example, in order toutilize an upper threshold of propionic acid during cultivation topromote OCFA production. Additionally, techniques and systems aredisclosed for identifying and using promotors of OCFA production.

In accordance with one or more embodiments of the present invention, amethod for incorporating microbial anaplerotic oil rich in odd-chainfatty acids (OCFA) into food for consumption is disclosed. The methodcomprises: identifying a unit amount of saturated fat that is present ina target food; determining an amount of microbial anaplerotic oil richin OCFA to incorporate into the target food, the determining comprising:identifying an amount of OCFA present in the microbial anaplerotic oilas a ratio of OCFA amount per unit of microbial anaplerotic oil, whereinat least five percent of the total fatty acid (TFA) of the microbialanaplerotic oil are OCFA; and combining the ratio with the unit amountof saturated fat resulting in the amount of microbial anaplerotic oilrich in OCFA to incorporate into the target food; and adding thedetermined amount of microbial anaplerotic oil rich in OCFA to thetarget food.

In accordance with one or more embodiments of the present invention, afood additive comprising microbial anaplerotic oil rich in odd-chainfatty acids (OCFA) is disclosed. The microbial anaplerotic oil isderived from a microbial strain cultured with added propionate resultingin elevated levels of OCFA in a microbial mass of the microbial strainwherein: at least five percent of the microbial anaplerotic oil from themicrobial strain's total fatty acid (TFA) content is odd-chain fattyacids (OCFA); and at least one percent cell dry weight (CDW) of themicrobial mass from the microbial strain is odd-chain fatty acids(OCFA).

In accordance with one or more embodiments of the present invention, afood rich in odd-chain fatty acids (OCFA) for consumption by human andnon-human animals is disclosed. The food comprises: an amount ofmicrobial anaplerotic oil derived from a microbial strain cultured withan added propionate precursor or added propionate, resulting in elevatedlevels of OCFA in a microbial mass, the microbial anaplerotic oilcomprising: a melting point greater than or equal to thirty-five degreesCelsius; at least five percent of the microbial anaplerotic oil from themicrobial strain's total fatty acid (TFA) content is odd-chain fattyacids (OCFA); and at least one percent cell dry weight (CDW) of themicrobial mass from the microbial strain is odd-chain fatty acids(OCFA).

To the accomplishment of the foregoing and related ends, the followingdescription and annexed drawings set forth certain illustrative aspectsand implementations. These are indicative of but a few of the variousways in which one or more aspects may be employed. Other aspects,advantages and novel features of the disclosure will become apparentfrom the following detailed description when considered in conjunctionwith the annexed drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The claimed matter may take physical form in certain parts andarrangements of parts, a preferred embodiment of which will be describedin detail in the specification and illustrated in the accompanyingdrawings which form a part hereof, and wherein:

FIG. 1 is a chromatogram of Aurantiochytrium acetophilum HS399displaying the microalgal fatty acid profile;

FIG. 2 is a line graph depicting the impact of propionatesupplementation on microalgae growth under batch and fed-batchconditions;

FIG. 3 is a flow chart of steps involved in a method according to anexemplary embodiment of the present disclosure;

FIG. 4 is a line graph depicting Aurantiochytrium acetophilum HS399growth in response to propionate supplementation;

FIG. 5 is a line graph depicting Aurantiochytrium acetophilum HS399residual glucose consumption in response to propionate supplementation;

FIG. 6 is a line graph depicting the culture pH-drift ofAurantiochytrium acetophilum HS399 fed with varying levels ofpropionate;

FIG. 7 is a line graph depicting Aurantiochytrium acetophilum HS399growth in response to propionate supplementation;

FIG. 8 is a line graph depicting Aurantiochytrium acetophilum HS399residual glucose consumption in response to propionate supplementation;

FIG. 9 is a line graph depicting the culture pH-drift ofAurantiochytrium acetophilum HS399 fed with varying levels ofpropionate;

FIG. 10 is a graph depicting fatty acid distribution throughout theculture of Aurantiochytrium acetophilum HS399 fed-batch at differentpropionic levels;

FIG. 11 is a graph depicting fatty acid accumulation throughout theculture of Aurantiochytrium acetophilum HS399 fed-batch at differentpropionic levels;

FIG. 12 is a line graph depicting OCFA accumulation in anAurantiochytrium acetophilum HS399 culture fed varying amounts ofpropionate;

FIG. 13 is a line graph depicting cell dry weight for anAurantiochytrium acetophilum HS399 culture fed varying amounts ofpropionate;

FIG. 14 is a line graph depicting Aurantiochytrium acetophilum HS399residual glucose consumption in response to propionate supplementation;

FIG. 15 is a line graph depicting the culture pH-drift ofAurantiochytrium acetophilum HS399 fed with varying levels ofpropionate;

FIG. 16 is a schematic diagram illustrating the active and passivetransport of propionic acid inside the cell. The pH gradient across thecell controls the passive uptake of propionic acid by the cell;

FIG. 17 is a line graph depicting the residual propionic acid as afunction of pH in a pH-auxostat culture of Aurantiochytrium acetophilum;

FIG. 18 is a line graph depicting dry cell weight for anAurantiochytrium acetophilum HS399 culture which shows the impact of pHand an organic acid feeding regime on HS399 growth;

FIG. 19 is a line graph depicting residual glucose as a function of thepH-set point in a pH-auxostat culture of Aurantiochytrium acetophilumHS399;

FIG. 20 is a line graph comparing the cell dry weight for anAurantiochytrium acetophilum HS399 culture fed propionic acid and anAurantiochytrium acetophilum HS399 culture that is not;

FIG. 21 is a line graph comparing the cumulative productivities for anAurantiochytrium acetophilum HS399 culture fed propionic acid and anAurantiochytrium acetophilum HS399 culture that is not;

FIG. 22 is a line graph depicting the residual glucose and ammonialevels;

FIG. 23 is a graph showing online monitoring of the fermenter HS399cultures with other parameters such as dissolved oxygen, feedstock andtitrant pumping rate, and pH control;

FIG. 24 is a line graph comparing the total propionate consumption of anAurantiochytrium acetophilum HS399 culture fed propionic acid and anAurantiochytrium acetophilum HS399 culture that is not;

FIG. 25 is a graphical representation of results of growth and substrateconsumption of Yarrowia lipolytica ATCC18944 using different carbonsources;

FIG. 26 is a micrograph illustrating the filamentous and yeastmorphology of Yarrowia lipolytica while producing OCFAs;

FIGS. 27 and 28 are graphical representations of cell dry weight,residual glycerol, and pH where Y. lipolytica is cultivated withincreasing daily propionate concentrations;

FIGS. 29 and 30 , are graphical representations of odd chain fatty acid% dry weight and total fatty acid % dry weight where Y. lipolytica iscultivated with increasing daily propionate concentrations;

FIG. 31 is a graphical representation of the A. acetophilum HS399 oxygenuptake (OUR) in response to pH driven propionate toxicity;

FIG. 32 is a 3D graphical representation of the propionic acid toxicityas cytosolic propionate is controlled by the extracellular pH andpropionate concentration;

FIG. 33 is a graphical representation of A. acetophilum HS399 in thepresence of batched or fed batch propionate at different dailyconcentrations;

FIG. 34 is a graphical representation illustrating results of growth ofAurantiochytrium acetophilum HS399 and residual ammonia when propionatewas fed in growth or lipid phase;

FIG. 35 is a graphical representation illustrating the growth of A.acetophilum HS399 with different carbon sources;

FIG. 36 is a graphical representation illustrating the residualpropionate cultures of A. acetophilum HS399 fed different carbonsources;

FIG. 37 is a graphical representation of example results ofsub-culturing A. acetophilum HS399 in a cyanocobalamin deprived mediafor over 10-generations;

FIG. 38 is a graphical representation of one implementation illustratingexample results of cell dry weight and residual propionate where theculture was initially fed 3 g/L propionate;

FIG. 39 is a graphical representation of the impact of cyanocobalamin inA. acetophilum HS399 growth and propionic acid consumption in 10 Lfermenters;

FIG. 40 is a graphical representation showing the impact of propionicacid exposure on A. acetophilum HS399 growth and odd chain fatty acidproduction in 10 L fermenters under two different growth modes;

FIG. 41 is a graphical representation illustrating results of oneimplementation, where, much like the two-stage growth mode describedabove, ammonia can be fed merely during the growth phase;

FIG. 42 is a graphical representation illustrating results of oneimplementation, applying the impact of ammonia to sodium hydroxide ratioof the feed in the total ammonia fed and biomass yields of a doubleauxostat culture of Aurantiochytrium acetophilum HS399;

FIG. 43 is a graphical representation illustrating results of oneimplementation, describing the impact of pH set-point control in thetransition of ammonia to propionic acid pH auxostat culture ofAurantiochytrium acetophilum HS399;

FIG. 44 is a graphical representation illustrating results forAurantiochytrium acetophilum HS399 double pH-auxostat cultures, showingthe impact of sodium hydroxide supplementation into the glucose fed inthe residual propionic acid control in a pH-auxostat;

FIG. 45 is a graphical representation showing cell dry weight andresidual glucose of A. acetophilum HS399 in a 100 L pilot fermenter;

FIG. 46 is a graphical representation of the cumulative productivity ofA. acetophilum HS399 in a 100 L pilot fermenter;

FIGS. 47A, 47B, 47C, 47D are graphical representations of online datareadings exhibited by A. acetophilum SH399 in a 100 L pilot fermenter;

FIG. 48 is a table, (Table 24) that illustrates total lipids and FattyAcid profile from a 1000 L pilot fermenter for Aurantiochytriumacetophilum HS399 odd chain fatty acid fermentation;

FIGS. 49A, 49B, 49C, 49D are graphical representations of A. acetophilumHS399 double pH-auxostat cultures for the production of odd chain fattyacids, growth productivity and lipid accumulation;

FIG. 50 is a graphical representation of online data readings of A.acetophilum HS399 double pH-auxostat cultures;

FIG. 51 is a graphical representation of an example result illustratinggrowth and residual propionate in A. acetophilum HS399 cultures that aresubject to propionic anaplerosis triggered by cyanocobalamin;

FIG. 52 is a graphical representation of residual glucose in A.acetophilum HS399 cultures subject to propionic anaplerosis triggered bycyanocobalamin;

FIG. 53 is a graphical representation showing growth inhibition of A.acetophilum HS399 by short chain fatty acids propionic acid (C3:0),pentanoic acid (C5:0) and heptanoic acid (C7:0);

FIG. 54 is a table (Table 25) that illustrates a list of results forrespective alternative promoters of odd chain fatty acid (OCFAs)production in Aurantiochytrium acetophilum HS399;

FIG. 55 is a table, (Table 26) that illustrates a list of fatty acidcompositions of several vegetable oils;

FIG. 56 is a graphical representation illustrating the growth ofAurantiochytrium acetophilum HS399 that has been subjected to propionicanaplerosis triggered by cyanocobalamin;

FIG. 57 is a graphical representation illustrating the residual glucoselevels in an Aurantiochytrium acetophilum HS399 culture that has beensubjected to propionic anaplerosis triggered by cyanocobalamin; and

FIG. 58 is a graphical representation illustrating the residualpropionate levels in an Aurantiochytrium acetophilum HS399 culture thathas been subjected to propionic anaplerosis triggered by cyanocobalamin.

DETAILED DESCRIPTION

The claimed subject matter is now described with reference to thedrawings, wherein like reference numerals are generally used to refer tolike elements throughout. In the following description, for purposes ofexplanation, numerous specific details are set forth in order to providea thorough understanding of the claimed subject matter. It may beevident, however, that the claimed subject matter may be practicedwithout these specific details. In other instances, structures anddevices are shown in block diagram form in order to facilitatedescribing the claimed subject matter.

With reference to the drawings, like reference numerals designateidentical or corresponding parts throughout the several views. However,the inclusion of like elements in different views does not mean a givenembodiment necessarily includes such elements or that all embodiments ofthe claimed subject matter include such elements. The examples andfigures are illustrative only and not meant to limit the claimed subjectmatter, which is measured by the scope and spirit of the claims.

Anaplerosis refers to the replenishment of citric acid intermediatesthat have been extracted by the cell for biosynthesis. Anapleroticsubstrates, such as glucose, protein and odd chain fatty acids (OCFAs),could be converted into citric acid intermediates to restore an energyimbalance of the cell. Anaplerotic substrates are often referred asgluconeogenic substrates. OCFAs are different from other anapleroticsubstrates because they can undergo ketosis and cross the blood-brainbarrier. Therefore, OCFAs have been associated with a decrease inmetabolic disease risk, and their intake has been proposed for thetreatment and prevention of various gene and brain disorders. Thepresence of OCFAs in diet is scarce and typically limited to ruminantfat (e.g., butter), which contains only trace amounts (<2% total fattyacid (TFA)) of pentadecanoic acid (C15:0) and heptadecanoic acid (17:0).Existing pharma OCFAs, such as tripentanoin and triheptanoin oils, areproduced synthetically, and are made of fatty acids that are nottypically present in a human diet. Alternatively, as described herein, aprocess may be devised that can result in a natural oil comprising large(e.g., >50 total fatty acid (TFA)) quantities of dietary (e.g., C15:0and C17:0) OCFAs.

Typical anaplerotic substrates can include pyruvate (e.g., derived fromcarbohydrates), glutamine/glutamate (e.g., derived from protein) andprecursors of propionyl-CoA, such as OCFAs. Anaplerotic substrates canbe used to restore energy balance in the mitochondria; and, there is awide range of pathologies to which OCFAs may provide benefits. As anexample, in this aspect, OCFAs have been experimentally used to treat:gene metabolic disorders, such as Glut1 deficiency, Fatty Acid OxidationDisorder (FAOD), Pyruvate Carboxylase Deficiency, CarnitinePalmitoyltransferase II Deficiency, Huntington, Phenylketonuria, AdultPolyglucosan Body Disease (APBD), and Long-Chain Fat OxidationDisorders; neural disorders, such as Epilepsy, Alzheimer's Disease, andAutism Spectrum Disorder (ASD); circulatory system disorders, anddiabetes type II and other diseases associated to the metabolic syndromeepidemics.

Dietary odd chain fatty acids (OCFA), pentadecanoic acid (C15:0) andheptadecanoic acid (C17:0), also known as margaric acid, may be derivedfrom ruminant fat (e.g., butter), and are thought to be likely derivedfrom bacterial anaerobic activity in the rumen of dairy producinganimals. These OCFAs can be found in very small amounts (e.g., <2% totalfatty acids (TFA)) in some dairy products (e.g., milk and butter).Pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) have also beenfound to be produced in the human gut, which may be triggered by dietaryfiber intake, presumably supporting bacterial anaerobic activity.Ref. 1. Because only trace amounts of odd chain fatty acids (e.g., C15:0and C17:0) are present in human diets, alternative sources (i.e.,nutraceuticals, medical foods or therapeutics) can be used tosignificantly increase the intake of this type of nutrient.

Currently, merely limited amounts of odd chain fatty acids (e.g., C15:0and C17:0) are readily available from known natural, dietary sources,such as ruminant fat. Techniques and systems can be devised forproducing a natural anaplerotic oil that contains significant dietaryOCFAs. In one aspect, compositions can be created that comprise a higherconcentration, than current sources, of odd chain fatty acids, such aspentadecanoic (C15:0) and heptadecanoic (C17:0) fatty acids. Further, inone aspect, a method can be devised for efficient and affectivegeneration of such fatty acids from a newly derived source.

Microbials can produce a variety of fatty acids, the composition ofwhich can vary among different strains. As an example, thraustochytridmicroalgae can accumulate lipids up to eighty-five (85%) of their dryweight; and, amongst the oleaginous microorganisms, they may be one ofthe fastest growing. Further, these organisms can be adapted tofermentation conditions (e.g., low shear sensitivity, highosmotolerance) for use in industrial production of microbe-based oils.For example, A. acetophilum HS399 is a thraustochytrid that can producean oil containing palmitic acid (e.g., 45% total fatty acids (TFA)), n-6docosapentaenoic acid (e.g., 8% TFA), and n-3 docosahexaenoic (e.g., 40%TFA) as the main fatty acids, with other fatty acids present in traceamounts. Pursuant to the requirements of the Budapest Treaty, a liveculture of the Aurantiochytrium acetophilum HS399 microalgae straindescribed herein was deposited on Sep. 12, 2019 at National Center forMarine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, EastBoothbay, Me. 04544, USA and received Bigelow Accession Number201909001.

The trace fatty acids of A. acetophilum HS399 can include pentadecanoicacid (C15:0) and heptadecanoic acid (C17:0) (e.g., at <0.3% TFA). Thetrace fatty acids, including these two identified fatty acids, aretypically ignored in the lipid profile reports for these organisms.OCFA, including pentadecanoic acid and heptadecanoic acid, are fattyacids that contain an odd number of carbon atoms in the structure. OCFAare typically related to bacterial activity (e.g., propionic acidbacteria), and are less likely to be present in algae, yeast/fungi, andplants.

FIG. 1 is a chromatogram of Aurantiochytrium acetophilum HS399illustrating the microalgae's fatty acid profile. As shown in FIG. 1 ,A. acetophilum HS399 naturally contains trace amounts of C15:0. Thepresence of trace amounts in A. acetophilum HS399 suggests that thepathway responsible for the synthesis of OCFA may be present in A.acetophilum HS399. Because of the composition of their fatty acidprofile, and their ability to be grown rapidly, microbials such as A.acetophilum HS399 may provide an attractive source of odd-chain fattyacids, by generating odd-chain fatty acids in a more concentrated mannerthan other known natural sources, such as milk fat (e.g., providing amore cost effective and efficient source of OCFA). As an example, abenefit of using microbials in place of butter and other ruminant fat isthe higher concentration of OCFA found in them. In addition, as anotherexample benefit, some microbial oil lacks residues of phytol or phytanicacid that are often present in ruminant fat. Consumption of phytol orphytanic acid can lead to health concerns in some individuals.

Techniques can be devised that provide for an increased production ofnaturally occurring odd-chain fatty acids from microbials than might begenerated from typical microbials. The cultivated microbial and/orisolated composition may be used individually as products or as aningredient in a variety of products. As an example, microalgae such asA. acetophilum HS399 can be cultivated to produce a desirable fatty acidprofile comprising OCFA, which may be isolated through variousextraction processes. In this example, the isolated oil from A.acetophilum HS399, containing the OCFA, may comprise a composition richin OCFA, such as pentadecanoic acid (C15:0) and heptadecanoic acid(C17:0). As described herein, in one implementation, the algae may becultivated using an improved method that includes the use of a complexculture media, which can promote increased production of the OCFA.

In one implementation, the microbials, such as microorganisms comprisingalgae, microalgae, yeast, and fungi, including species from the classLabyrinthulomycetes, such as the species Aurantiochytrium acetophilum,may be cultivated using an improved method that includes the presence ofa complex media, which can promote increased production of the OCFA. Inthis implementation, an amount of heptadecanoic acid produced by A.acetophilum HS399 can increase from <0.3 up to 1% TFA when a complexculture media containing yeast extract and proteose peptone is used as areplacement for previously utilized media (e.g., defined media). In thisimplementation, the increase of heptadecanoic acid may be proportionalto the amount of proteose peptone used in the complex culture media. Theincrease in heptadecanoic acid in the cultured A. acetophilum HS399,using this technique, suggests that the presence of OCFA inthraustochytrids may not be strain specific, nor stress related. Ref. 3,5. Instead, using this technique, the increase in heptadecanoic acid islikely due to the presence of added nutrients in the media that providefor the accumulation of this OCFA. Therefore, the high levels of oddchain fatty acids reported in some thraustochytrids strains (Ref. 4)might not be a strain specific trait, nor a physiological response tostress (Ref. 3,5) but rather the result of poor growth and/or high yeastextract or other OCFA precursors.

In one implementation, propionic acid (e.g., and or one or morepropionates, such as the anion, salts, and/or esters of propionic acid)may be used as a precursor for production of OCFA. Proteose peptonecomprises valine, isoleucine, and methionine amino acids, respectivelycomprising at least a three-carbon chain, which may provide a precursorthree-carbon backbone of propionic acid. In this implementation, forexample, it is likely that A. acetophilum HS399 can incorporatepropionic acid in its lipid generation pathway, resulting in theproduction of OCFA.

Generally, fatty acid synthesis in oleaginous microbials consists of alipid synthesis pathway involving acetyl CoA, and some metabolic cycles.As an example, acetyl-coenzyme A (Acetyl-CoA) is a universal two carbondonor, or building block, for fatty acid biosynthesis. Acetyl-CoA can besupplied by multiple paths, from various origins, and then subsequentlyactivated into acetyl-acyl carrier protein (ACP) or converted to MalonylCoA through Acetyl-CoA carboxylase. Later, by sequential reactions ofcondensation, reduction dehydration and reduction, palmitic acid will beproduced.

In one aspect, analysis of the genome of A. acetophilum HS399 suggeststhat saturated fats are synthesized through the Fatty Acid Synthase(FAS) pathway that uses acetyl-coA as a building block for the fattyacid elongation. The production of even chain fatty acids uses amalonyl-ACP as a substrate for elongation. As described herein, in oneimplementation, when propionic acid is present the acyl carrier protein(ACP) cleaves to methyl-malonyl instead of malonyl, resulting in the FASproducing of odd chain fatty acids instead of even chain fatty acids.Palmitic acid (C16:0) is typically the primary even chain fatty acid inA. acetophilum HS399, while the primary OCFA is typically pentadecanoic(C15:0) instead of heptadecanoic acid (17:0). In this implementation,fatty acid synthesis of palmitic acid (C16:0) undergoes through6-consecutive elongation cycles, while the (C15:0) OCFA undergoes only5-elongation cycles before the fatty acid is liberated from the acylcarrier protein. This suggests that FAS is governed by a chain lengthfactor.

In one aspect, propionic acid is commonly used for its antimicrobialcharacteristics, among other things. For example, propionic acid caninhibit growth of mold and bacteria at low levels (e.g., <1% by weight),and is often used as an antimicrobial agent to preserve animal and/orhuman food sources. Other uses include adding propionic acid to productsto mitigate algae growth on surfaces. In this aspect, as an illustrativeexample, FIG. 2 illustrates propionic acid's growth inhibitorycharacteristics for A. acetophilum HS399, at concentrations as low as 3grams/liter (g/L); and lethality to A. acetophilum HS399 atconcentrations of 10 g/L.

In this aspect, in addition to the common and traditional use ofpropionic acid as an antimicrobial agent that kills algae, as describedherein, techniques have been devised for propionic acid to be used tofacilitate in growing algae, and/or to increase OCFA production in thealgae. In one implementation, in this aspect, propionic acid (e.g.,and/or propionates) can be introduced into an algal bioprocess using afed-batch approach, while reducing the potential toxic effects on thealgae. FIG. 3 is a flow diagram illustrating an exemplary method 300 forintroducing propionic acid into an algal growth culture program. Theexemplary method begins at 302. At reference numeral 304, amicroorganism (e.g., microalgae such as A. acetophilum HS399) can beadded to the culture medium. At reference numeral 306, propionic acidmay be added to the culture medium comprising the microorganism (e.g.,A. acetophilum HS399) in a batch, continuous or fed-batch process, andcultured in a bioreactor having with the culture medium (e.g., organic).

In one embodiment, the propionic acid can be added at a ratio of atleast 0.05 g of propionic acid per gram of A. acetophilum HS399 biomass,in order to accumulate elevated amounts of OCFA. In one embodiment, 0.15g of propionic acid per gram of A. acetophilum HS399 biomass, in orderto accumulate OCFA above 50% TFA. In another implementation, thepropionic acid can be added at a rate of above zero and up to about 3g/L per day. In one implementation, the propionic acid can be added at arate of above zero and up to about 3 g/L per day for three days,resulting in a total propionic acid addition of about 9 g/L. In oneembodiment, adding the propionate can comprise adding the propionate ina fed-batch into the culture medium. In one embodiment, adding thepropionate can comprise adjusting the propionate fed to produce OCFAs ina range of 5 and 70% TFAs.

At reference numeral 308, anaplerotic oil containing concentratedamounts of OCFA can be extracted from the A. acetophilum HS399. In oneembodiment, anaplerotic oil can be produced from the culturedmicroorganisms, wherein at least five percent of the total fatty acids(TFA) of the anaplerotic oil are OCFA, and OCFA make up at least onepercent cell dry weight (CDW) of the anaplerotic oil. Having extractedthe anaplerotic oil containing concentrated amounts of OCFA theexemplary method 300 ends at 310.

In one implementation, the propionate fed approach can cause somemicroorganism (e.g., A. acetophilum HS399) growth inhibition, forexample, but may not result in a complete culture loss of themicroorganism batch. In this implementation, the fed-batch approach canachieve similar cell densities and overall lipid accumulation as asimilar control batch with no propionic acid fed, with merely a one-daydifference. As one example, propionic acid can be fed into the algalculture batch on demand (e.g., automatically, using a pH-auxostat fedbatch system). As another example, propionic acid can be fed into thealgal batch, along with a carbon source (e.g., glucose, glycerol oracetate), at a ratio below 0.1 of weight to weight (w/w) of propionicacid to carbon source (propionic acid/carbon source). In anotherexample, propionic acid can be fed along with the carbon source at aratio below 0.05 w/w propionic acid/carbon source, to mitigate avoidaccumulation of propionate in the culture media. In one example,propionic acid may be fed into a culture at a culture pH higher than 5.A low pH increases the toxicity of propionic acid making it moredifficult to balance the window between propionate incorporation andgrown inhibition.

EXAMPLES

Embodiments described herein are exemplified and additional embodimentsare disclosed in further detail in the following Examples, which are notin any way intended to limit the scope of any aspects of the inventiveconcepts, described herein.

Example 1—Propionic Acid Incorporation into A. acetophilum Fatty AcidSynthase (FAS)

In one example implementation, the resulting impact on growth and lipidaccumulation of Aurantiochytrium acetophilum HS399 when using propionicacid can be illustrated. In this implementation, four treatments can beprepared with varying concentrations of propionate (e.g., 0, 10, 20, 30g/L), hereinafter: “P0, P10, P20 and P30” respectively. In thisimplementation, propionate can be batched as sodium propionate in aflask culture. Respective Erlenmeyer flasks (250 mL) can be inoculated(1% v/v) in triplicates with a 24 hour (h) old culture of A. acetophilumHS399 and incubated in an orbital shaker at 180 rpm and 27° C.

In this implementation, the respective Erlenmeyer flasks contain 100 mLof a medium supplemented with (g/L): dextrose (50), ammonium acetate(2.3), NaCl (12.5), MgSO₄ 7H₂O (2.5), KH₂PO₄ (0.5), KCl (0.5) and CaCl₂)(0.1). This medium also contains trace element solution (5 ml/L) andvitamin solution (1 ml/L). The trace element solution contains (g/L):EDTA di-sodium salt (6), FeCl₃ 6H₂O (0.29), H₂BO₃ (6.84), MnCl₂ 4H₂O(0.86), ZnCl₂ (0.06), CoCl₂ 6H₂O (0.026), NiSO₄ 6H₂O (0.052), CuSO₄ 5H₂O(0.002), Na₂MoO₄ H₂O (0.005). The vitamin solution contains (mg/L):thiamine (100), biotin (0.5) and cyanocobalamin (0.5). All culturematerials can be autoclaved (121° C., 15 min) and media can be filtersterilized before use. A propionic acid stock solution (200 g/L) can beused to feed propionic acid. Daily samples can be collected to analyzethe cell dry weight, residual glucose, culture pH, lipid and fatty acidcomposition of the cultures. Cell dry weights are analyzed by vacuumfiltration (0.2 μm) and washed with a solution of ammonium bicarbonate.Residual glucose is analyzed using a colorimetric method based onglucose peroxidase activity. Biomass for lipid analysis can becentrifuged and washed using purified water. The washed biomass can befreeze dried. Total lipids are analyzed using the Folch method (AOAC996.06) and the FAMES are analyzed by gas chromatography usingnonadecanoic (C19:0) acid as an internal standard.

FIG. 4 illustrates the resulting cell dry weights, and the resultingresidual glucose is illustrated shown in FIG. 5 . As illustrated by theresults, propionic acid is lethal at concentrations of 20 g/L and 30g/L, while concentrations of 10 g/L are strongly inhibitory to thegrowth of A. acetophilum HS399. FIG. 6 illustrates the A. acetophilumHS399 growth results in the alkalization of the medium, presumablyassociated to the consumption of organic acids. As shown by the lipidand fatty acid data at 68 h of incubation from P0 and P10 in Table 1below, the presence of propionate decreases lipid and total fatty acidaccumulation. Further, the presence of propionate produces results in adecrease in palmitic (C16:0) and an increase in saturated odd chainfatty acids (OCFAs) tridecanoic (C13:0), pentadecanoic (C15:0) andheptadecanoic acid (C17:0), which can result in propionicincorporation/deposition (see Table 2). As illustrated, the increase oftotal OCFA (C13, C15, C17) from 0.2% at zero propionate to 63% totalOCFA at 10 g/L propionate suggests that propionate is incorporated intothe fatty acid synthase pathway (FAS). The lack of OCFA in thepolyunsaturated fraction suggests that propionate may not beincorporated/deposited in the polyketide synthase pathway (PKS).

TABLE 1 Impact of propionate supplementation in HS399 lipid and fattyacid profile after 68 h incubation Propionate (g/L) 0 10 Total Lipids (%DW) 83.0 ± 0.0  55.3 ± 0.6  Total Fatty Acids (% DW) 69.5 ± 0.6  42.8 ±0.9  Fatty Acid Profile (% TFA) 13:0 0.0 ± 0.0 3.0 ± 0.1 14:0 3.1 ± 0.00.9 ± 0.0 15:0 0.2 ± 0.0 51.7 ± 0.6  16:0 52.7 ± 0.1  5.1 ± 0.3 17:0 0.0± 0.0 8.3 ± 0.0 18:0 1.6 ± 0.0 0.2 ± 0.1 22:5 (n-6) 7.3 ± 0.1 3.0 ± 0.122:6 (n-3) 32.6 ± 0.1  24.9 ± 0.4  Other FA 2.2 ± 0.0 2.2 ± 1.2 OCFA (%TFA) 0.2 ± 0.0 63.1 ± 0.7 

TABLE 2 Propionate deposition, feeding rate and productivity of OCFAs inresponse to propionate supplementation PA Feeding Propionate OCFAPropionate Rate deposition Productivity (g/L) (g_(PA)/g_(Biomass)) (%)(g/L/d) 0 0.00 ± 0.00 0.0 ± 0.0 0.00 ± 0.00 1100 1.12 ± 0.05 6.9 ± 0.50.81 ± 0.06

Example 2—Propionic Concentration in Aurantiochytrium Fatty Acid Profile

In another implementation, fed-batching Aurantiochytrium acetophilumHS399 with propionic acid may impact its growth, and lipid accumulation.As an example, in this implementation, four treatments can be preparedwith varying concentrations of propionate (0, 3, 6, 9 g/L), hereinafter“P0, P3, P6 and P9” respectively. Propionate can be fed-batch fed assodium propionate in a flask culture with daily additions at 3 g/L.Respective Erlenmeyer flasks (250 mL) are inoculated (1% v/v) intriplicates with a 24 h old culture of A. acetophilum HS399. Thecultures are incubated in an orbital shaker at 180 rpm and 27° C.

Respective Erlenmeyer flasks contain 100 mL of a medium supplementedwith (g/L): dextrose (100), ammonium acetate (4.6), NaCl (12.5), MgSO₄7H₂O (2.5), KH₂PO₄ (0.5), KCl (0.5) and CaCl₂) (0.1). This medium alsocontains trace element solution (5 ml/L) and vitamin solution (1 ml/L).The trace element solution contains (g/L): EDTA di-sodium salt (6),FeCl₃ 6H₂O (0.29), H₂BO₃ (6.84), MnCl₂ 4H₂O (0.86), ZnCl₂ (0.06), CoCl₂6H₂O (0.026), NiSO₄ 6H₂O (0.052), CuSO₄ 5H₂O (0.002), Na₂MoO₄ H₂O(0.005). The vitamin solution contains (mg/L): thiamine (100), biotin(0.5) and cyanocobalamin (0.5). All culture materials can be autoclaved(e.g., 121° C., 15 min) and the media can be filter sterilized beforeuse. A propionic acid stock solution (200 g/L) can be used as the fedpropionic acid. In this example, P0 is not fed any propionate, P3 is fed3 g/L on day 0 (inoculation day), P6 is fed 3 g/L on day 0 and 3 g/L onday 1, and P9 is fed 3 g/L on day 0, 1 and 2. Daily samples arecollected to analyze the cell dry weight, residual glucose, culture pH(see FIG. 9 ), and lipid and fatty acid composition of the cultures.Cell dry weights (CDW) can be analyzed by filtration (e.g., 0.2 μmfilter media) using a vacuum, and washed with a solution of ammoniumbicarbonate. Residual glucose can be analyzed using colorimetric methodsbased on glucose peroxidase activity. Biomass for lipid analysis iscentrifuged and washed using purified water. The washed biomass isfreeze dried. Total lipids are analyzed using Folch method (AOAC 996.06)and the FAMES re analyzed by gas chromatography using nonadecanoic(C19:0) acid as an internal standard.

In this example, as shown by the cell dry weights represented in FIG. 7, and the residual glucose represented in FIG. 8 , the treatment thatutilized propionic acid produced results illustrating growth inhibitionat concentrations as low as 3 g/L (P3). Further, these resultsillustrate that the growth inhibition effect may be dose dependent, withthe strongest inhibition resulting from treatments having higherpropionate concentrations (P6 and P9). Even though growth inhibition wasexhibited, the 70 h growth achieved in this example for the fed batchingof 9 g/L of propionate (˜15 g/L) was higher than the growth achieved forthe of batching 9 g/L of propionate at inoculation (see Example 1). Thisexample result illustrates that fed-batching can be an effectivestrategy for mitigating propionic toxicity, while inducing the cells toproduce OCFAs.

As an example of this strategy, the lipid and fatty acid data isrepresented in Tables 3-5, below. These results illustrate lipidaccumulation observed at 68 h, 96 h, and 116 h of incubation,respectively. As shown in these Tables, the differences in lipidaccumulation due to propionate toxicity decreased at 96 h and 116 h,from the 68 h observation. As illustrated, the treatments, includingthose supplemented with propionate, accumulated lipids above 70% DW evenwith growth inhibition. Results of this example illustrate that higher,desired amount of odd chain fatty acids can be accumulated at 96 h forboth P6 (62.4% TFA) and P9 (also 62.4% TFA), which illustrates that the0.18 g of propionate per gram of biomass supplied in P6 may beappropriate to improve OCFA accumulation in A. acetophilum HS399 to adesirable level.

As an example, adding more propionic acid to these treatments/culturesmay not further increase the propionic acid, but may increase propionictoxicity with potentially negative impact in growth and lipidaccumulation. Further, as an example, adding less propionate (e.g., 0.6g propionate per gram of biomass), may result in low OCFAs accumulationdue to palmitic acid synthesis taking over the OCFAs synthesis after 68h, as shown in FIG. 10 , FIG. 11 and FIG. 12 , and summarized in Table6, below. In this example, the high (e.g., ˜66%) propionic aciddeposition in fatty acids suggest that propionate may be incorporated(e.g., at least once) in each fatty acid, presumably in the firstcondensation step of the FAS pathway (methyl malonyl acyl carrierprotein condensation). In this example, the remaining propionate may beoxidized, and lost through anaplerosis into the Citric Acid Cycle.

TABLE 3 Lipid and fatty acid profile at 68 h Propionate (g/L) 0 3 3 + 33 + 3 + 3 Total Fatty 66.5 ± 1.5  45.1 ± 3.0  44.5 ± 4.5  40.7 ± 1.7 Acids (% DW) Fatty Acid Profile (% TFA) 13:0 0.0 ± 0.0 3.8 ± 0.6 5.2 ±0.2 5.5 ± 0.5 14:0 4.0 ± 0.1 2.0 ± 0.3 1.5 ± 0.1 1.2 ± 0.0 15:0 0.2 ±0.0 44.7 ± 6.0  49.6 ± 0.6  48.8 ± 0.1  16:0 47.1 ± 0.1  10.8 ± 5.6  5.4± 0.7 5.0 ± 0.0 17:0 0.0 ± 0.0 5.3 ± 0.6 5.8 ± 0.2 5.7 ± 0.2 18:0 1.5 ±0.0 0.2 ± 0.3 0.0 ± 0.0 0.0 ± 0.0 22:5 (n-6) 8.6 ± 0.0 4.5 ± 0.5 3.6 ±0.1 3.5 ± 0.1 22:6 (n-3) 36.9 ± 0.3  26.3 ± 0.7  25.7 ± 0.1  26.8 ± 0.3 Other FA 2.2 ± 0.2 2.8 ± 0.2 3.3 ± 0.1 3.7 ± 0.0 OCFA 0.2 ± 0.0 52.9 ±7.3  60.9 ± 0.6  60.6 ± 0.4  (% TFA)

TABLE 4 Lipid and fatty acid profile at 96 h Propionate (g/L) 0 3 3 + 33 + 3 + 3 Total Lipids 82.3 ± 1.2  81.0 ± 1.0 73.3 ± 1.5  70.0 ± 1.0  (%DW) Total Fatty Acids 70.6 ± 3.7  66.8 ± 4.8 62.5 ± 0.3  56.3 ± 1.2  (%DW) Fatty Acid Profile (% TFA) 13:0 0.0 ± 0.0  1.4 ± 0.1 4.1 ± 0.1 4.4 ±0.1 14:0 3.8 ± 0.1  2.8 ± 0.0 1.2 ± 0.1 1.1 ± 0.0 15:0 0.2 ± 0.0 19.7 ±0.8 51.9 ± 0.2  51.7 ± 0.2  16:0 47.1 ± 0.4  33.9 ± 0.6 4.3 ± 0.2 3.8 ±0.1 17:0 0.0 ± 0.0  2.7 ± 0.0 6.4 ± 0.1 6.3 ± 0.0 18:0 1.5 ± 0.0  1.0 ±0.0 0.1 ± 0.0 0.1 ± 0.0 22:5 (n-6) 8.6 ± 0.1  6.6 ± 0.1 3.5 ± 0.1 3.4 ±0.1 22:6 (n-3) 36.5 ± 0.4  29.4 ± 0.2 24.6 ± 0.1  24.9 ± 0.1  Other FA2.1 ± 0.0  2.2 ± 0.1 3.4 ± 0.2 3.6 ± 0.1 OCFA (% TFA) 0.2 ± 0.0 24.0 ±1.0 62.8 ± 0.2  62.9 ± 0.2 

TABLE 5 Lipid and fatty acid profile at 116 h Propionate (g/L) 0 3 3 + 33 + 3 + 3 Total Lipids (% 82.0 ± 1.7  74.8 ± 0.3 77.7 ± 0.6  74.0 ± 1.0 DW) Total Fatty Acids 70.7 ± 2.2  71.7 ± 4.3 70.8 ± 0.6  63.1 ± 2.0  (%DW) Fatty Acid Profile (% TFA) 13:0 0.0 ± 0.0  1.1 ± 0.0 2.7 ± 0.1 3.0 ±0.1 14:0 3.7 ± 0.1  2.5 ± 0.0 1.3 ± 0.0 1.1 ± 0.0 15:0 0.3 ± 0.1 18.6 ±0.6 46.0 ± 1.1  49.2 ± 0.3  16:0 46.7 ± 0.1  33.8 ± 0.5 8.7 ± 1.0 5.8 ±0.3 17:0 0.0 ± 0.0  3.0 ± 0.1 7.7 ± 0.2 8.2 ± 0.1 18:0 1.5 ± 0.0  1.1 ±0.0 0.2 ± 0.0 0.2 ± 0.0 22:5 (n-6) 8.7 ± 0.1  6.8 ± 0.0 4.4 ± 0.2 4.0 ±0.0 22:6 (n-3) 36.8 ± 0.3  30.4 ± 0.0 25.6 ± 0.2  24.9 ± 0.1  Other FA2.2 ± 0.2  2.8 ± 0.2 3.3 ± 0.1 3.7 ± 0.0 OCFA (% TFA) 0.3 ± 0.1 22.8 ±0.8 57.5 ± 1.4  60.8 ± 0.2 

TABLE 6 Results of propionate feeding regime on propionate depositionand productivity of OCFAs by Aurantiochytrium acetophilum HS399 PAFeeding Propionate OCFA Propionate Rate deposition Productivity (g/L)(g_(PA)/g_(Biomass)) (%) (g/L/d) 0 (96 h) 0.00 ± 0.00  0.0 ± 0.0 0.0 ±0.0 3 (96 h) 0.08 ± 0.00 57.0 ± 0.9 1.53 ± 0.02 3 + 3 (116 h) 0.18 ±0.00 65.0 ± 0.6 2.89 ± 0.06 3 + 3 + 3 (166 h) 0.28 ± 0.00 39.5 ± 1.72.56 ± 0.04

Table 5 (above) provides results of varying use of propionic acid in analgal culture after 116 hours culturing. As an example, the results inTable 5 illustrate four different approaches to the use of propionicacid in a batch of Aurantiochytrium acetophilum HS399, as indicated bythe four columns: 0, 3, 3+3, and 3+3+3. The first column indicates nouse of propionic acid in the algal batch; the second column indicatesthe use of merely one does of 3 g/L of propionic acid in the algalbatch, the third column indicates the use of two separate doses of 3 g/Leach of propionic acid, on separate days; and the fourth columnindicates the use of three separate doses of 3 g/L each of propionicacid, at one per day. The respective rows of the Table 5 are indicativeof the resulting percentage dry weight (% DW) levels of total lipids,total fatty acids, and each fatty acid profile for the respectiveapproaches (e.g., titled by the fatty acid name or indicator, such asC13:0 (13 chain FA), C14:0 (14 chain fatty acid), etc.).

As illustrated, the use of propionic acid (in columns 2, 3 and 4)indicates an increase in the presence of pentadecanoic acid (C15:0:fifteen-chain FA) in the resulting Aurantiochytrium acetophilum HS399batch, in a dose response manner. As illustrated in column one, no useof propionic acid results in 0.3% pentadecanoic acid of the TFA content.Column two shows that the addition of 3 g/L of propionic acid results inabout 18% (18.6%) of pentadecanoic acid of the TFA content; column threeshows the batch addition of two separate doses of 3 g/L of propionicacid results in about 40% (46%) of pentadecanoic acid of the TFAcontent; and column four shows the batch addition of three separatedoses of 3 g/L of propionic acid (e.g., such as over a three-day period)results in greater than 40% (49.2%) of pentadecanoic acid of the TFAcontent.

Conversely, as seen in Table 5, the addition of the propionic acidindicates a reduction in the resulting palmitic acid (C16:0 or e.g.,palmitate, such as the salts and esters of palmitic acid) over the samefour dose approaches. That is for example, in Table 5, the palmitic acid(C16:0) indicates 46.7% of the TFA profile with no propionic acid; 33.8%at one 3 g/L dose; 8.7% at two doses of 3 g/L each; and 5.8% at threeequal doses of 3 g/L. These results suggest that the number of iterativeelongation cycles in fatty acid synthetase pathway may be determined bythe length of the acyl chain. That is, in the presence of propionicacid, the fatty acid synthetase can prefer to eliminate one elongationcycle and produce pentadecanoic (C15:0), rather than producingheptadecanoic acid (C17:0), through full-seven-elongation cycles. Thisresult appears to be consistent with the hypothesis that stearic acid(C18:0) is not a direct product of fatty acid synthetase, but theelongation of palmitic acid (C16:0).

Further, as illustrated in Table 5, the use of propionic acid in thealgal culture batch can also result in production of other fatty acids,such as heptadecanoic acid (C17:0) and tridecanoic acid (C13:0) (e.g.,both odd-chain fatty acids), with the total OCFA indicating a resultabove 60% of TFA (60.4% of TFA total for C13:0+C15:0+C17:0). Asindicated in Table 5, as the OCFA production increases, in therespective columns 2-4, the amount of resulting palmitic acid is reducedto 5.8% of the TFA. Additionally, results indicate that the amount ofdocosahexaenoic acid (DHA) also decreases to greater than about 20%(24.9%) of the TFA. This result may suggest that propionic acid enhancesthe synthesis of saturated fats from the fatty acid synthase (FAS) overthe production of polyunsaturated fatty acids through the polyketidesynthase (PKS) pathway.

In one aspect, the resulting product of an algal culture batch utilizingthe multi-step propionic dose approach (e.g., three doses of 3 g/L eachover three days) may be a highly concentrated anaplerotic oil frommicroalgae. That is, for example, the resulting product can compriseabout 38% of the cell dry weight (CDW) of OCFAs (e.g., 60.4% OCFAs ofthe 63.1% TFA=38.1% OCFAs of total DW of algal product). In this aspect,no other natural source (e.g., non-synthetic) is known to produce thesequantities or concentrations of odd chain fatty acids per batch product(e.g., >50% TFA; and >30% CDW).

In this implementation, as shown in the pentadecanoic (15:0) row ofTable 5, the disclosed process can increase the pentadecanoic acidcontent from about 0.3% TFA, in the resulting product, without the useof propionate, to about 49% in a fed-batch culture with 0.15 gpropionate per gram of biomass. In one implementation, it may bedesirable to control the daily propionic feed in order to controltoxicity (as described herein); however, a per gram biomass fedpropionate may be more desirable than the g/L measurement forcontrolling the product OCFA composition. The per gram biomass fedmetric could be translated to different reactors, while the g/Lmeasurement may be reactor or process specific.

In this example, as shown in Table 5, using this same comparison,heptadecanoic acid (C17) content results are shown to increase from justa trace amount to greater than about 5% (8%) TFA. Further, in thisimplementation, while the concentration of DHA (C22:6) is shown todecrease from about 37% to about 25%, the presence of DHA in theresulting biomass may comprise a significant source of this oil, whencompared to synthetically produced anaplerotic oils (e.g., containingodd-chain fatty acids that can improve anaplerotic conditions), whichtypically lack DHA entirely.

That is, for example, tripentanoin and triheptanoin (short andmedium-sized odd-chain fatty acids) are currently the primaryconcentrated sources of odd chain fatty acids available. However, thesemolecules are produced synthetically and do not resemble any naturallyavailable oil. For example, the odd-chain triheptanoin does not existnaturally, and is obtained through chemical synthesis from glycerol andheptanoic acid (C7:0). In contrast, algal anaplerotic oil can beproduced naturally, as described above; and the resulting oils cancontain the same odd chain fatty acids that are present in dairyproducts (e.g., and other natural sources), for example, which may allowfor appropriate introduction into a human diet. As an example, theanaplerotic oil production, described herein, can be from algae sources,and is believed to be less costly than synthetic production because itdoes not utilize modified fatty acids and chemical transesterification.

Example 3—Propionic Acid can Inhibit Growth at Various Concentrations

As another example, a strategy can be devised for propionate feeding toAurantiochytrium acetophilum HS399 cultures, and the results canillustrate the effect on growth and propionate deposition in the lipidfraction. In this example, five treatments (P0, P3, P0+3, P0+1+1+1,P0+2+2+2) can be used, and respective treatments can receive a differentamount of propionate, which is dependent on incubation time. In thisexample, treatment P3 receives 3 g/L propionate on day 0 at thebeginning of the protein phase, while P0+3 receives 3 g/L propionate onday 1 at the beginning of the end of the protein phase-beginning of thelipogenic phase. The treatment P0+1+1+1 receives 1 g/L propionate on day1, 2 and 3, and treatment P0+2+2+2 receives 2 g/L on day 1, 2 and 3 inan attempt to reduce the high residual propionate available duringlipogenesis. Respective Erlenmeyer flasks (250 mL) are inoculated (1%v/v) in triplicates with a 24 h old culture of A. acetophilum HS399 andincubated in an orbital shaker at 180 rpm and 27° C.

Respective Erlenmeyer flasks contain 100 mL of a medium supplementedwith (g/L): dextrose (100), ammonium acetate (4.6), NaCl (12.5), MgSO₄7H₂O (2.5), KH₂PO₄ (0.5), KCl (0.5) and CaCl₂ (0.1). This medium alsocontains trace element solution (5 ml/L) and vitamin solution (1 ml/L).The trace element solution contains (g/L): EDTA di-sodium salt (6),FeCl₃ 6H₂O (0.29), H₂BO₃ (6.84), MnCl₂ 4H₂O (0.86), ZnCl₂ (0.06), CoCl₂6H₂O (0.026), NiSO₄ 6H₂O (0.052), CuSO₄ 5H₂O (0.002), Na₂MoO₄ H₂O(0.005). The vitamin solution contains (mg/L): thiamine (100), biotin(0.5) and cyanocobalamin (0.5).

In this example, respective culture materials are autoclaved (e.g., 121°C., 15 min) and the media is filter sterilized before use. A propionicacid stock solution (200 g/L) can be used as the fed propionic acid.Daily samples are collected to analyze the cell dry weight, residualglucose, culture pH (see FIG. 15 ), and lipid and fatty acid compositionof the cultures. Cell dry weights are analyzed by filtration (e.g., 0.2μm filter media) using a vacuum and washed with a solution of ammoniumbicarbonate. Residual glucose is analyzed using a colorimetric methodbased on glucose peroxidase activity. Biomass for lipid analysis iscentrifuged and washed using purified water. The washed biomass isfreeze dried. Total lipids are analyzed using Folch method (AOAC 996.06)and the FAMES are analyzed by gas chromatography and flame ionizationdetection using nonadecanoic (C19:0) acid as an internal standard.

As shown in FIGS. 13 and 14 respectively, the resulting cell dry weightand resulting residual glucose illustrate that feeding 3 g/L propionicacid (P3) provides a lag in growth in the first 24 h. Further, theresults illustrate that this lag was mitigated by feeding 3 g/L at theend of the protein phase-beginning of lipogenesis (day 1). Additionally,the results illustrate that initial (24 h) growth for P0+3 was similarto the growth of the control treatment P0, and the growth lag observedin the P3 treatment was mitigated.

Table 7, below, provides the lipid and fatty acid analysis results, andTable 8 provides the propionate deposition. As shown in these tables,postponing the propionic feed to the lipogenic phase (P0+3) resulted ina higher production of OCFAs (27.4 TFA) and higher propionate deposition(59.3±1.1% of total) than when feeding the propionate initially (P3), atthe beginning of the protein phase (25.2% TFA and 52.2±0.7% of total).Further, the results suggest that propionic acid lost through itsoxidation by the citric acid cycle can be higher during the proteinphase than the lipid phase. As an example, this may demonstrate thatwaiting for lipogenesis to feed propionate can help to mitigate toxicityand can help improve propionate incorporation into the OCFAs.Additionally, the results illustrate that OCFAs acid productivity mayincrease by approximately 20% by feeding propionate merely duringlipogenesis (from 1.18±0.02 at P3 to 1.18±0.02 g OCFAs/L d for P0+3).

As an example, results suggest that fractionating the 3 g/L ofpropionate into three daily dosage of 1 g/L (P0+3 vs. P0+1+1+1) may notreduce the impact of propionic acid toxicity in A. acetophilum HS399growth, as illustrated in FIG. 13 . In this example, residualconcentrations as low a 1 g/L may provide some growth inhibition.Further, the results obtained with P0+2+2+2, suggest that 6 gpropionate/L produces a desirable amount (e.g., higher) of OCFAs in A.acetophilum HS399 flask cultures, which may translate into 0.18 g ofpropionate per g of biomass produced for other growth platforms.

TABLE 7 Total lipids and fatty acid profile at time of harvest (96 h)Propionate (g/L) 0 + 1 + 0 + 2 + 0 3 0 + 3 1 + 1 2 + 2 Harvest & SampleDay 4 6 6 6 6 Total Lipids (% DW) 79.3 ± 0.6  78.0 ± 0.0 79.5 ± 0.5 79.7 ± 0.6 76.0 ± 1.0  Ash (% DW) Total Fatty Acids (% DW) 69.7 ± 1.4 64.8 ± 1.2 66.0 ± 2.2  67.2 ± 5.0 63.5 ± 1.6  Fatty Acid Profile (% TFA)13:0 0.0 ± 0.0  1.1 ± 0.0 1.0 ± 0.0  1.1 ± 0.0 1.8 ± 0.0 14:0 4.1 ± 0.1 2.3 ± 0.0 2.2 ± 0.1  2.0 ± 0.0 1.1 ± 0.0 15:0 0.2 ± 0.0 20.1 ± 0.2 22.1± 0.2  23.6 ± 0.4 42.3 ± 0.7  16:0 47.1 ± 0.4  31.3 ± 0.2 29.1 ±0.3 27.1 ± 0.2 7.6 ± 0.2 17:0 0.0 ± 0.0  4.0 ± 0.0 4.3 ± 0.1  4.6 ± 0.1 8.8± 0.1 18:0 1.4 ± 0.0  1.1 ± 0.0 1.0 ± 0.0  0.9 ± 0.0 0.2 ± 0.0 22:5(n-6) 8.1 ± 0.0  6.1 ± 0.0 5.8 ± 0.1  5.8 ± 0.0 4.2 ± 0.2 22:6 (n-3)36.5 ± 0.3  30.9 ± 0.2 31.5 ± 0.4  31.9 ± 0.2 30.1 ± 0.4  Other FA 2.3 ±0.1  2.7 ± 0.1 2.6 ± 0.0  2.7 ± 0.1 3.3 ± 0.1 OCFA (% TFA) 0.4 ± 0.025.5 ± 0.2 27.6 ± 0.1  29.2 ± 0.4 53.5 ± 0.8 

TABLE 8 Impact of propionate feeding regime on propionate deposition andproductivity of OCFAs at 120 h incubation. PA Feeding Propionate OCFAPropionate Rate deposition Productivity (g/L) (g_(PA)/g_(Biomass)) (%)(g/L/d) 0 (Day 4) 0.00 ± 0.00  0.0 ± 0.0 0.0 ± 0.0 3 (Day 5) 0.09 ± 0.0052.2 ± 0.7 1.04 ± 0.01 0 + 3 (Day 5) 0.09 ± 0.00 59.3 ± 1.1 1.18 ± 0.020 + 1 + 1 + 1 (Day 5) 0.09 ± 0.00 59.6 ± 1.4 1.19 ± 0.03 0 + 2 + 2 + 2(Day 5) 0.20 ± 0.00 46.7 ± 0.9 1.86 ± 0.04

Example 4—Modeling Propionic Acid Toxicity

In one aspect, the uptake activity of propionate as an organic carbonsource by microalgae may be dependent on the culture's pH. For example,when propionic acid is fed to a culture that is growing at a pH of 7,the residual organic acid can be mostly dissociated in the propionateform (propionic pKa=4.88), with only a minor amount remainingundissociated. In this aspect, while the propionate and free proton formenters the microalgae cell through a monocarboxylic symport structure,propionic acid is membrane permeable and may be diffused directly intothe microalgae cell. Therefore, in this aspect, the uptake of propionatecan be controlled by the cell, and the uptake of propionic acid may notbe controlled by the cell. As an example, the uncontrolled uptake ofpropionic acid presumably lowers the internal Aurantiochytrium cell pH;in turn, the cell attempts to maintain its pH homeostasis by pumpingprotons out of the cell. In this example, a build-up of propionateinside the cell can result, which is proportional to the pH gradientbetween the intracellular and the extracellular (see FIG. 16 ).Therefore, in one implementation in this aspect, the internal propionateconcentration, which could be used to measure propionic cell toxicity,may be calculated using at least the external culture pH, the internalcell pH, and the residual acetate concentration in the culture. In oneimplementation, the change in pH may be calculated with the followingequation derived from the Henderson and Hasselbalch equation:

${\Delta{pH}} = {{{pH}_{i} - {pH}_{o}} = {\log\left( \frac{\left\lbrack P_{i} \right\rbrack}{\left\lbrack P_{o} \right\rbrack} \right)}}$

In this implementation, pHi=pH inside the cell, pHo=pH outside the cell,P=propionate, O=outside cell, I=inside cell. Assumptions made in thiscalculation may include: the impact of the ionic strength in thepropionic acid dissociation constant as negligible, propionic acid butnot propionate is membrane permeable, and there are no other protectionmechanisms involved in the regulation of cell pH. The relationship maybe illustrated with the non-limiting Example 1 (above), which shows thatpropionate may be substantially lethal at residual (extracellular)concentrations of ˜15 g/L and a pH of 6. The above-mentioned model canbe used to translate this Example 1 into an intracellular propionateconcentration of 176 g/L, providing a value that could be translated atdifferent pHs and residual concentrations. Further, the relationship maybe illustrated by the non-limiting Example 2 (above), which shows thatpropionate may be substantially non-lethal, but growth inhibitory, atconcentrations as low as 1 g/L at pH of 7. The above-mentioned model canbe used to translate Example 2 into an intracellular propionateconcentration of 1 g/L, providing a value that could be translated atdifferent pHs and residual concentrations. Therefore, in thisimplementation, the model can help provide an understanding of theimpact of medium pH in propionate toxicity and can provide a tool tomodel propionic acid toxicity that could be used to improve a process tooptimize OCFAs production.

Example 5—Propionic Acid/pH-Auxostat LED Strategy to Produce OCFA

In one implementation, a propionic acid/pH-auxostat led strategy may beused to produce anaplerotic oils containing odd chain fatty acids usingthe microalgae A. acetophilum HS399. In this implementation, forexample, three treatments can be fed propionic acid as titrant tomaintain pH at a desired level (e.g., organic acids: Propionic acid-pH7,Propionic acid-pH6 and Propionic acid-pH5). Further, a control treatmentcan be fed acetic acid at pH 7 (e.g., Acetic acid-pH7). In thisimplementation, initially (24 h incubation), the culture pH may not becontrolled, and the pH of the respective treatments can drift from 7.5to 8. After 24 h incubation, the desired pH set point of each treatmentcan be set using the respective organic acids.

In this implementation, for example, bubble column reactors (1.3 L) areinoculated (1% v/v) in triplicates with a 24 h old culture of A.acetophilum HS399. The cultures are aerated at 1.4 vvm and maintained at27° C. under axenic conditions. The bubble columns contain 700 mL of amedium supplemented with (g/L): dextrose (40), ammonium acetate (1.1),NaCl (12.5), MgSO₄ 7H₂O (2.5), KH₂PO₄ (0.5), KCl (0.5) and CaCl₂ (0.1).This medium also contains a trace element solution (5 ml/L) and avitamin solution (1 ml/L). The trace element solution can contain (g/L):EDTA di-sodium salt (6), FeCl₃ 6H₂O (0.29), H₂BO₃ (6.84), MnCl₂ 4H₂O(0.86), ZnCl₂ (0.06), CoCl₂ 6H₂O (0.026), NiSO₄ 6H₂O (0.052), CuSO₄ 5H₂O(0.002), Na₂MoO₄ H₂O (0.005). The vitamin solution can contain (mg/L):thiamine (100), biotin (0.5) and cyanocobalamin (0.5).

The respective culture materials can be autoclaved (121° C., 15 min) andthe media can be filter sterilized before use. In this example,propionic and acetic acid are diluted to 3% w/w and added to the acidcontainer, from which the cultures are fed through a peristaltic pump inresponse to the pH drift above their set point. Daily samples can becollected to analyze the cell dry weight, residual glucose, culture pH,and lipid and fatty acid composition of the cultures. Cell dry weightsare analyzed by filtration (0.2 μm filter media) using a vacuum andwashed with a solution of ammonium bicarbonate. Residual glucose isanalyzed from culture supernatant (5000 g; 5 min) using a colorimetricmethod based on glucose peroxidase activity. Residual acetate andpropionate are analyzed by HPLC using external standard. Biomass forlipid analysis can be centrifuged and washed using purified water; andthe washed biomass is freeze dried. Total lipids are analyzed usingFolch method (AOAC 996.06), and the FAMEs are analyzed by gaschromatography and flame ionization detection using nonadecanoic (C19:0)acid as an internal standard.

Residual propionate results are provided in FIG. 17 , illustrating thatthe pH-auxostat strategy was able to successfully maintain thepropionate levels at the pH 5 and 6 set points. Further, as illustratedin FIG. 17 , the treatments comprising a pH 7 set point did not appearto have sufficient residual propionate or acetate to identify whetherthe organic acid was present during the demonstration, and thereforethey were not fed on demand, but ad libitum. The results of this exampleillustrate that using a propionic acid-pH7 pH-auxostat system may haveinterrupted the propionic acid feeding due to the displacement ofresidual propionate. As illustrated by the cell dry weight datarepresented in FIG. 18 , propionic acid was not growth inhibitory.However, as illustrated by the data in Table 9, below, OCFA (6.3% TFA)may not be accumulated to levels observed previously in the flask (>50%TFA) or at lower pH treatments, likely due to the lack of propionicacid. Therefore, in one implementation, at a higher pH, supplementationwith sodium propionate, or target alkalization of the media, may be usedto provide for the presence of residual propionate in the batch even athigh pH-set points. These results indicate that the lower pH set pointscan be highly growth inhibitory, as shown by the cell dry weight data(FIG. 18 ) and glucose analyses (FIG. 19 ). These results indicate thatinhibition may be higher at lower pHs, as may be predicted by the modeldescribed in Example 4 (Table 10 below).

TABLE 9 Lipid and fatty acids analyses from the pH auxostat Aceticacid-pH 7 Propionic acid-pH 7 Propionic acid-pH 6 Propionic acid-pH 5Consumed 0.0 ± 0.0  1.0 ± 0.1   2.4 ± 0.1   2.3 ± 0.2  Propionate (g/L)Propionic Acid 0.0 ± 0.0 24.7 ± 1.0  40.3 ± 1.6  30.9 ± 2.0  Deposited(%) Total Lipids 89.5 ± 0.5  87.5 ± 1.5  79.5 ± 1.5  75.0 ± 3.0  (% CDW)Total Fatty Acid 71.9 ± 5.2  70.3 ± 2.8  63.4 ± 0.3  59.2 ± 3.1  (% CDW)Fatty Acid (% TFA) (% DW) (% TFA) (% DW) (% TFA) (% DW) (% TFA) (% DW)C11:0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 0.1 ± 0.1 0.0 ±0.0 0.0 ± 0.0 C13:0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 2.2 ± 0.31.4 ± 0.2 2.1 ± 0.1 1.2 ± 0.0 C14:0 3.2 ± 0.0 2.3 ± 0.2 2.9 ± 0.1 2.0 ±0.1 1.3 ± 0.0 0.8 ± 0.0 1.1 ± 0.1 0.7 ± 0.1 C15:0 0.0 ± 0.0 0.0 ± 0.07.3 ± 1.7 5.1 ± 1.0 50.6 ± 0.3  32.1 ± 0.0  51.6 ± 1.7  30.5 ± 0.6 C16:0 56.4 ± 0.0  40.6 ± 2.9  49.3 ± 1.7  34.7 ± 2.6  9.6 ± 0.8 6.1 ±0.5 8.1 ± 1.8 4.8 ± 1.3 C17:0 0.0 ± 0.0 0.0 ± 0.0 1.7 ± 0.4 1.2 ± 0.27.2 ± 0.4 4.6 ± 0.3 7.3 ± 0.1 4.3 ± 0.3 C18:0 1.3 ± 0.0 1.0 ± 0.1 1.1 ±0.0 0.8 ± 0.1 0.3 ± 0.0 0.2 ± 0.0 0.1 ± 0.1 0.1 ± 0.1 C20:3n6 & 0.2 ±0.0 0.2 ± 0.0 0.1 ± 0.1 0.1 ± 0.1 0.2 ± 0.2 0.1 ± 0.1 0.3 ± 0.0 0.2 ±0.0 C21:0 C20:5n3 & 0.4 ± 0.0 0.3 ± 0.0 0.4 ± 0.0 0.3 ± 0.0 0.4 ± 0.00.3 ± 0.0 0.5 ± 0.0 0.3 ± 0.0 C22:0 C22:5n6 DPA 6.7 ± 0.0 4.8 ± 0.4 6.5± 0.0 4.6 ± 0.2 4.7 ± 0.0 3.0 ± 0.0 4.4 ± 0.2 2.6 ± 0.2 C22:6n3 31.6 ±0.0  22.7 ± 1.6  30.7 ± 0.1  21.6 ± 0.9  23.4 ± 0.4  14.8 ± 0.2  24.7 ±0.6  14.6 ± 0.4  OCFA (% TFA) 0.0 ± 0.0 0.0 ± 0.0 9.0 ± 2.1 6.3 ± 1.260.0 ± 0.2  38.0 ± 0.1  60.9 ± 1.7  36.0 ± 0.9 

TABLE 10 The intracellular propionate, represented as an indicator ofpropionate toxicity for each treatment, was calculated from thepropionate residual concentration and the pH set point using modeldescribed in Example 4. Actual Values Theoretical Values AverageResidual D1-D4 Average Internal Propionic Acid (g/L) Propionic Acid(g/L) Acetic acid-pH 7 0.0 ± 0.0 0.00 Propionic acid-pH 7 0.1 ± 0.1 0.13Propionic acid-pH 6 0.4 ± 0.1 4.18 Propionic acid-pH 5 0.6 ± 0.2 56.70

Example 6—Single Stage Approach to Producing OCFA

In another implementation, a New Brunswick 10 L Bioflo Pro 300 fermenter(Eppendorf) can be used to provide an improvement in the productivity ofOCFAs accumulation, previously identified in flasks and bubble columns.In this implementation, a bioprocess can be devised to incorporate oneor more portions of one or more techniques described herein. In thisimplementation, a propionic acid/pH-auxostat strategy can be adoptedafter day 1 to induce the production of OCFAs. As an example, in thisimplementation, the pH-auxostat is operated at a pH set point of 6.5 tomitigate propionic toxicity. The pH-auxostat is activated through theaddition of 0.5 g/L of potassium propionate to provide residualpropionate availability during the culture. A control treatment can beprovided with propionate, to be used as comparison. Fermenterscontaining 5 L of fresh media are inoculated (1% v/v) in triplicateswith a 24 h old culture of A. acetophilum HS399. The fermenters areaerated at 0.5 vvm and are maintained at 27° C. under axenic conditions.A stirring speed is increased in response to identified dissolved oxygenvalues below 10% saturation, from 200 rpm up to 1000 rpm.

In this implementation, for example, the 5 L of batch media can contain(g/L): corn syrup D95 (31), ammonium acetate (19.5), MgSO₄ 7H₂O (2.5),KH₂PO₄ (2.5), KCl (1) and CaCl₂) (0.2). This medium can also contain atrace element solution (25 ml/L) and vitamin solution (5 ml/L). Thetrace element solution contains (g/L): EDTA di-sodium salt (6), FeCl₃6H₂O (0.29), H₂BO₃ (6.84), MnCl₂ 4H₂O (0.86), ZnCl₂ (0.06), CoCl₂ 6H₂O(0.026), NiSO₄ 6H₂O (0.052), CuSO₄ 5H₂O (0.002), Na₂MoO₄ H₂O (0.005);and the vitamin solution contains (mg/L): thiamine (100), biotin (0.5)and cyanocobalamin (0.5). In this example, the fermenter is fed another5-6 L of a medium containing (g/L): ammonium phosphate (2.5), ammoniumhydroxide (29% pure) (15.2), corn syrup DE95 (1143). This medium canalso contain a trace element solution (25 ml/L) and a vitamin solution(5 ml/L).

In this example, this medium is fed in a DO-stat mode in response todissolved oxygen values detected above 15% saturation. As an example,the dissolved oxygen values can trigger a feeding pulse of 0.3 ml/L minthat lasts 102 min. The pH is maintained at 5.8 using NaOH, while thebatch ammonia is consumed. The pH can drift to higher values when theresidual ammonia is substantially exhausted from the fermenter. Atsubstantial residual ammonia exhaustion, the pH can be controlled withpropionic acid at a pH 6.4, in some examples, while the pH of thecontrol treatment may drift up to 7-8 without titration. Culturematerials and media can be autoclaved (e.g., 121° C., 15 min) whileseparating the nitrogen and the carbon source. Foam can be controlled(e.g., automatically) through the addition of (<1 ml/L) Hodag antifoam.

In this implementation, samples can be collected daily to analyze thecell dry weight, residual glucose, culture pH, and lipid and fatty acidcomposition of the cultures. Cell dry weights can be analyzed byfiltration (e.g., 0.2 μm filter media) using a vacuum, and washed with asolution of ammonium bicarbonate. Residual glucose can be analyzed fromculture supernatant (e.g., 5000 g; 5 min) using a colorimetric methodbased on glucose peroxidase activity. Residual acetate and propionatecan be analyzed by HPLC using an acceptable external standard. Thebiomass for lipid analysis can be centrifuged and washed using purifiedwater, and the washed biomass can be freeze dried. Total lipids areanalyzed using Folch method (AOAC 996.06) and the FAMEs are analyzed bygas chromatography and flame ionization detection using nonadecanoic(C19:0) acid as an internal standard.

Example results of the cell dry weights, in this implementation, areshown in FIG. 20 . As an example, the results illustrate that, eventhough the propionic acid titration slightly inhibited A. acetophilumHS399 growth compared to no propionic acid titration, the culturespresented 170 g/L cell dry weight, and accumulated lipids at 70% DW,with 60% TFA being OCFAs. The results illustrated in FIG. 21 show thatthe average cumulative productivities are approximately 30 g/L/d, whichtranslates into 10 g/L/day of OCFAs. In this implementation, asillustrated by these results, the example bioprocess can maintain theresidual glucose and ammonia at desired levels, as illustrated by FIG.22 , and other cultivation parameters at desired levels, as shown inFIG. 23 . As illustrated in FIG. 24 , providing results of totalpropionate consumed during the process in this implementation, theprocess provided a consumption of 0.155 g of propionic per gram ofbiomass, which is in agreement with the values observed in flask(Example 2). In this implementation, as in previous implementations, theresults of the OCFAs produced by the control treatment were negligible.

Fungi/Yeast

In another aspect, techniques and systems can be devised, as describedherein, for a natural method of improving the production of OCFAs inmicroalgae and other microorganisms, like yeast/fungi, that do notutilize genetic modification. A process is disclosed herein for theproduction of an oil rich in OCFA. As one example, oil rich in OCFA maybe produced at 28 g oil/L/day, and at 31 g/L/day or more. For example,such processes can produce a triacylglyceride containing OCFAs of 40% ofTFA or more, and up to approximately 67% TFA. In one example, thecultures may achieve a final biomass yield of approximately 126 g/L.

In addition to A. acetophilum HS399 microalgae, as described above,certain microorganisms (e.g., yeast/fungus) can produce a variety offatty acids, the composition of which can vary among different strainsof microorganisms. As an example, Yarrowia lipolytica is considered tobe an oleaginous yeast that can accumulate large amounts of lipids.Yeast species are typically described as “oleaginous” if the lipids theyaccumulate account for more than 20% of their biomass. For Y.lipolytica, the amount of lipid accumulation is dependent on the strainand the carbon source, along with growth conditions. Under optimalgrowth conditions, some fed-batch cultures of Y. lipolytica can store43% lipids of their cell dry weight (CDW) in continuous fermentationsusing industrial glycerol and may store up to 54% lipids of their CDW inbatch cultures on a stearin-based medium. In these examples, most of thelipids accumulating in Y. lipolytica are triacylglycerols rather thanfree fatty acids (FFA), with C16 and C18 compounds being the mostabundant, with other fatty acids present in trace amounts.

The trace fatty acids of Y. lipolytica can include pentadecanoic acid(C15:0) and heptadecenoic acid (C17:1 n−8) (e.g., at <0.3% TFA). Thetrace fatty acids, including these two identified fatty acids, aretypically ignored in the lipid profile reports for these organisms.Odd-chain fatty acids (OCFAs), including pentadecanoic acid andheptadecenoic acid (C17:1 n−8) are fatty acids that contain an oddnumber of carbon atoms in the structure. OCFAs are typically related tobacterial activity (e.g., propionic acid bacteria), and are less likelyto be present in other microbes, such as yeast/fungi and microalgae, orplants.

The presence of trace amounts of OCFA in yeast/fungi suggests that thepathway responsible for the synthesis of OCFA may be present inyeast/fungi. Because of the composition of their fatty acid profile, andyeast/fungi ability to be grown rapidly, yeast/fungi such as Y.lipolytica may provide an attractive source of OCFA. Such microorganismsmay be able to generate OCFA in a more rapid and concentrated mannerthan other known natural sources, such as milk fat (e.g., providing amore cost effective and efficient source of OCFAs). Yeast/fungi may alsoprovide alternative OCFA, such as heptadecenoic acid (17:1 n−8), whichmay not be found in other food sources. Further, for example, yeast canproduce OCFAs without highly unsaturated fatty acids, which can helpmitigate undesirable flavors often associated with this type of oil. Asan example, a benefit of using yeast/fungi in place of butter and otherruminant fat is the higher concentration of OCFA found in these types ofyeast/fungi. In addition, as another example, benefit, yeast/fungi oillacks residues of phytol or phytanic acid that are often present inruminant fat. Consumption of phytol or phytanic acid can lead to healthconcerns in some individuals.

In one aspect, techniques can be devised that provide for an increasedproduction of naturally occurring odd-chain fatty acids from yeast/fungithan might be generated from typical yeast/fungi. The resultingcultivated yeast/fungi and/or resulting isolated composition may be usedindividually as products or as an ingredient in a variety of products.As an example, yeast/fungi such as Y. lipolytica can be cultivated andproduce a desirable fatty acid profile comprising OCFAs, which may beisolated through various extraction processes. In this example, theisolated oil containing the OCFAs may comprise a composition rich inOCFAs, such as pentadecanoic acid (C15:0) and heptadecenoic acid (C17:1n−8). In one implementation, in this aspect, the yeast/fungi may becultivated using an improved method that includes the presence of acomplex media, which can promote increased production of the OCFAs. Asone example, the cultivation media may comprise propionate. Propionateis a conjugate base of propionic acid and is a short-chain fatty acidoften produced by gut flora in some animals.

In one implementation, Y. lipolytica can be cultivated with propionateto increase the amount of OCFA production. The following describesexample implementations:

Example 7—Production of OCFA in Yarrowia lipolytica

Methods

Culture conditions. In one implementation, Yarrowia lipolytica ATCC18944can be cultivated in triplicates (n=3) in a medium containing (g/L):glycerol (80), monosodium glutamate (5), yeast extract (1), NaCl (12.5),MgSO₄ (7), H₂O (2.5), KCl (0.5), CaCl₂ (0.1), KH₂PO₄ (0.5), trace metalsolution (5 mL) and vitamin solution (1 mL). The trace element solutionmay contain (g/L): EDTA di-sodium salt (6), FeCl₃ 6H₂O (0.29), H₂BO₃(6.84), MnCl₂ 4H₂O (0.86), ZnCl₂ (0.06), CoCl₂ 6H₂O (0.026), NiSO₄ 6H₂O(0.052), CuSO₄ 5H₂O (0.002), Na₂MoO₄ 2H₂O (0.005). The vitamin solutioncan be filter-sterilized (e.g., using 0.2 μm pore size filter)containing (mg L-1): thiamine (100), biotin (0.5) and cyanocobalamin(0.5) unless otherwise stated. In some implementations cyanocobalaminmight be added to or subtracted from the medium formulation to change orimpact propionic acid deposition in OCFA. In some implementations cobaltcan be reduced or eliminated from the media to avoid synthesis ofcyanocobalamin that could compromise propionic acid deposition. In oneimplementation, a concentration of the cyanocobalamin and/or cobalt inthe culture medium can be below 0.4 μM.

Y. lipolytica cultures can be inoculated at 1% v/v in a 250 mL baffledErlenmeyer flask containing 100 mL of the above-mentioned media. Theflasks can be incubated in the dark in an orbital shaker at 180 rpm, 27°C. In this implementation, sodium propionate is fed batch every dayaccording to respective treatments. As an example, treatment0+0+2+2+2+2, is fed 2 g/L propionic acid at 48, 72, 96 and 120 hours,while treatment 0 is not fed any propionate at all.

Analyses. Cell dry weights can be obtained by drying samples that arepreviously vacuum filtrated (e.g., using 0.2 μm pore size filters).Residual propionate is analyzed directly by high performance liquidchromatography (HPLC). Lipids and total fatty acids are analyzed using adirect extraction, transesterification followed by gas chromatography(GC) and detection by flame ionization (FID). The different fatty acidsare identified and quantified using appropriate internal and externalstandards.

Propionic acid deposition rate. Propionate consumption can be calculatedbased on the residual propionate on day 0 and subtracting the finalresidual propionate, to find the consumption amount. The totalpropionate deposited in the biomass is calculated based on the finalcell dry weight, multiplied by the total fatty acid (TFA) ratio inbiomass, multiplied by the OCFA ratio in TFA, and multiplied by themolar factor of propionate in odd chain fatty acid, averaged at 0.3. Thepropionate deposition rate can be calculated by dividing the propionatedeposited by the propionate consumed and expressed as percent.

Results & Discussion

In this implementation, it is desirable to determine the capacity ofYarrowia lipolytica ATCC18944 to produce odd chain fatty acids (OCFAs)though the incorporation of propionate in the media. For example,propionate could be either incorporated in the lipids as OCFAs oroxidized through either the methylmalonate or methylcitrate pathways.Testing showed that the incorporation of medium propionic acid into Y.lipolytica fatty acids (i.e., OCFAs) may not be affected by the presenceor absence of cyanocobalamin (see Table 11 below). Cyanocobalamin isco-factor on the methylmalonate pathway, thus the results indicate thatthis pathway might not be active in Y. lipolytica.

TABLE 11 Impact of cyanocobalamin in propionic acid deposition inYarrowia lipolytica ATCC18944. Cyanocobalamin (μM) 0.00037 0 Dailypropionate (g/L d) 0 + 0 + 2 + 2 + 2 + 2 0 + 0 + 2 + 2 + 2 + 2 Cell dryweight (g/L) 5.3 6.0 Consumed propionate 1.7 2.6 (g/L) Odd chain fattyacids 0.9 1.4 (% DW) Odd chain fatty acids 0.05 0.1 (g/L) Propionate(MW)/ 0.3 0.3 OCFA (MW) Propionate 0.83 0.99 deposition (%)

In this aspect, for example, the methylcitrate pathway is the only knownanaplerotic pathway involved in propionic acid catabolism. FIG. 25 is agraphical representation 2500 of results of growth and substrateconsumption of Yarrowia lipolytica ATCC18944 using different carbonsources. As illustrated in FIG. 25 , Y. lipolytica may be able to growon propionate 2502 as sole carbon source. Therefore, as an example,because anaplerosis is used to sustain the growth on propionate as asole carbon source, these results suggest that propionate is primarilyoxidized through the methylcitrate pathway. Yarrowia lipolytica growthon propionate 2502 is shown to be much lower than on glucose 2504, butthis trend was corrected when both substrates 2506 (propionate andglycerol) were fed simultaneously.

In some implementations, fed batch propionate in the presence ofglycerol may be used to increase cell growth rates and OCFAs production.As one example, as illustrated in Tables 12 and 13, below, for Y.lipolytica, propionic acid deposition rates in OCFAs may be relativelylow (e.g., <3% TFA), which indicates that methylcitrate may readilyoxidize propionic acid. In one implementation, in order to mitigate thepropionic acid oxidative loss, the total amount of propionate fed batchcan be increased gradually from 0.3 to 8 g/L, which can result in13.3±1.3 TFA, 2.2±0.2% DW and 0.33 g/L OCFAs (see Table 13 below). Themain OCFAs produced by Yarrowia lipolytica were the Omega-8heptadecenoic acid (C17:1 n−8) and pentadecanoic acid (C15:0).

TABLE 12 Lipid and fatty acid analyses of Yarrowia lipolytica ATCC18944fed increasing daily propionate concentrations. Time (hrs) 0 h 168 168168 Daily propionate (g/L) Initial 0 0.3 + 0.3 0 + 0.6 Total Lipids (%DW) 24.7 ± 0.6 23.7 ± 3.5 21.7 ± 3.2 Total Fatty Acids 10.6 24.0 ± 1.521.7 ± 2.5 21.3 ± 2.8 (% DW) Fatty Acid Profile (% TFA) 15:0 0.00  0.3 ±0.0  0.3 ± 0.0  0.4 ± 0.1 16:0 11.10 10.8 ± 0.1 11.6 ± 0.6 12.3 ± 0.816:1 15.16 14.1 ± 0.1 14.2 ± 0.4 14.4 ± 0.3 17:1 0.00  1.1 ± 0.0  1.9 ±0.3  2.1 ± 0.5 18:0 4.75  4.8 ± 0.1  5.0 ± 0.3  5.3 ± 0.4 18:1 (n-9)56.59 54.9 ± 0.3 54.2 ± 1.4 52.0 ± 2.4 18:2 (n-6) 0.00 12.0 ± 0.4 10.8 ±0.7 11.3 ± 0.5 Other FA 12.40  1.4 ± 0.1  1.5 ± 0.2  1.6 ± 0.3 OCFA (%DW) 0  0.3 ± 0.03  0.5 ± 0.02  0.5 ± 0.09 OCFA (% TFA) 0  1.4 ± 0.04 2.3 ± 0.30  2.6 ± 0.83

TABLE 13 Lipid and fatty acid analyses of Yarrowia lipolytica ATCC18944fed increasing daily propionate concentrations. Time (h) 0 h 168 h 168 h168 h Daily Propionate (g/L) 0 + 0 + 1 + 0 + 0 + 2 + Initial 0 1 + 1 + 12 + 2 + 2 Total Lipids 10.00 22.7 ± 1.2 19.0 ± 0.0 18.3 ± 0.6 (% DW)Total Fatty Acids 7.9 22.0 ± 1.8 17.3 ± 1.6 16.2 ± 0.5 (% DW) Fatty AcidProfile (% TFA) 15:0 0.0  0.3 ± 0.0  1.3 ± 0.0  1.4 ± 0.1 16:0 14.0 12.4± 0.5 13.2 ± 1.0 13.8 ± 1.6 16:1 15.7 13.8 ± 0.4 12.2 ± 0.1 12.0 ± 1.117:1 0.0  1.1 ± 0.0 10.0 ± 0.7 10.0 ± 1.2 18:0 6.4  5.2 ± 0.3  6.3 ± 0.7 6.9 ± 1.4 18:1 (n-9) 49.5 52.0 ± 0.5 40.0 ± 0.0 38.8 ± 0.4 18:2 (n-6)14.4 13.9 ± 0.2 14.2 ± 0.7 13.1 ± 0.6 Other FA 0.0 61.3 ± 0.8  2.7 ± 0.554.0 ± 3.0 OCFA (% FA) 0  1.4 ± 0.1 13.1 ± 0.6 13.3 ± 1.3 OCFA (% DW)0.0  0.3 ± 0.0  2.3 ± 0.3  2.2 ± 0.2

As an example, unlike A. acetophilum HS399, Yarrowia lipolytica canaccumulate OCFA without accumulating Docosahexaenoic acid (DHA), whichmight be beneficial for certain formulations when long chainpolyunsaturated fatty acids (e.g., like DHA) are not desired. Asillustrated in FIG. 26 , in one implementation, Y. lipolytica canproduce OCFAs in polymorphic cultures 2600, which are combinations offilamentous molds and yeast morphology. As an example, these resultssuggest that, in addition to production in algae (e.g.,Aurantiochytrium), OCFAs may also be produced by fungi, regardless oftheir yeast or filamentous morphology.

FIGS. 27 and 28 are graphical representations of implementations whereY. lipolytica is cultivated with increasing daily propionateconcentrations 2700, 2800. In these implementations, the resultsillustrate that growth of the Y. lipolytica may not be affected by up to2 g/L d propionic fed despite the low pH (3) present. As illustrated,under these conditions the accumulation of cytoplasmic propionateequates to 328 g/L, which indicates how this strain can have a hightolerance to propionic acid toxicity.

Further, FIGS. 29 and 30 , are graphical representations ofimplementations where Y. lipolytica is cultivated with increasing dailypropionate concentrations 2900, 3000. In these examples, the resultsillustrate that the Y. lipolytica cells can accumulate lipids above 20%DW, with OCFA concentrations above 10% of TFA. As an example, therelatively low OCFAs levels produced by Y. lipolytica may be associatedwith the catabolic loss of propionate through the methylcitrate pathway.In one implementation, to address this issue, and to increase thepropionic acid incorporation in OCFAs, certain supplements may be addedto the media during propagation to mitigate the flow of the propionatethough the methylcitrate pathway, which can help mitigate propionic acidoxidative loss. As an example, itaconic acid can be used as a potentialinhibitor of the methyl isocitrate enzyme. Table 14, below indicatesthat itaconic acid may not always provide for mitigation of thepropionic acid depositions. One or more alternative inhibitor, such as3-Nitropropionate, 3-bromo pyruvate and V-13-009920, may provideimproved mitigation of the loss of propionate to the methylcitratepathway.

TABLE 14 Propionic acid deposition in the presence of itaconic acidItaconic acid (g/L)/Propionate (g/L) Time (h) 192 0/6 0.5/6 Cell dryweight (g/L) 8.3 ± 1.2 7.9 ± 1.1 Consumed propionate (g/L) 5.1 ± 1.5 5.0± 1.6 Propionate (MW)/OCFA (MW) 0.3 0.3 Odd chain fatty acids (% DW) 1.3± 0.5 1.1 ± 0.6 Propionic acid deposition (%) 0.6 ± 0.1 0.5 ± 0.2

In summary, in this implementation, Yarrowia lipolytica could be used toproduce oils rich in OCFAs. The OCFA concentration in the resulting oil,and the productivity, may be below that obtained with the microalgae A.acetophilum HS399. However, Y. lipolytica provides advantages becausethe main OCFA is C17:1 n−8, and Y. lipolytica does not produce highlyunsaturated fatty acids like may be found with A. acetophilum HS399,which might be preferred in certain applications.

Example 8—Propionate Induced Growth Inhibition

In another aspect, techniques may be devised to determine intracellularpropionate accumulation in microbials. In this aspect, a potentiallylethal concentration of propionate may be identified for desiredcultures of OCFA producing biologicals that are utilizing propionate toincrease OCFA production. In this way, for example, a high thresholdamount of propionate may be identified for desired biologicals thatproduce desired OCFA accumulation while maintaining a desired growthrate for the target biologicals.

In one implementation, the model used to determine intracellularpropionate accumulation, using Henderson and Hasselbach equations, canbe calibrated by establishing the lethal concentration threshold ofintracellular propionate that A. acetophilum (HS399) could tolerate. Thecalibration can be further verified using two different approaches,propionate concentration and pH-driven propionic acid toxicity.

In a first implementation, an Erlenmeyer flask can be inoculated with 0,10, 20, and 30 g/L initial treatments of propionate, at a substantiallyconstant pH of 6.4. In this implementation, observation indicates thatthe treatments containing 20 and 30 g/L will not survive. This model canbe used to translate the extracellular pH and propionic acidconcentration to intracellular propionate concentrations, a metric thatcould be interpreted across different pH values and propionateconcentrations scenarios. Table 15 below illustrates that initialpropionate treatment of propionate at 20 g/L results in lethality of theculture. Further, the cytosolic (the cytoplasmic matrix is the liquidfound inside cells) propionate concentration is identified to be 97.7g/L using the model.

TABLE 15 Propionic acid toxicity lethal threshold measured according totwo different approaches. Toxicity Applied pH Drift Initial PropionateToxicity Type Acute Chronic Metric Used OUR CDW pH 5.3 6.4 PropionateTreatment (g/L)  2.3 ± 0.1 20 Cytosolic Propionate (g/L) 107.5 ± 3.597.7

In a second implementation, the evolution of the oxygen uptake rate(OUR) is measured for two cultures where the decreasing pH set point iscontrolled with propionic acid. FIG. 31 is a graphical representation ofan example 3100 where, in this implementation, the pH 3102 can besteadily decreased from 7 down to 4 at a rate of 0.7 pH units per hour3104. In this example 3100, during the four hours of pH ramp, residualpropionate concentration in the media is maintained relatively constant(e.g., 2.3-2.4 g/L). In this implementation, the OUR remains relativelyconstant until the pH reached 5.3, and the OUR 3106 measurement droppedin response to propionate toxicity. In FIG. 31 , the example 3100 showsmonitoring of A. acetophilum HS399 oxygen uptake rate (OUR) in responseto pH driven propionate toxicity. In this example, the resultsillustrate that cell respiration was not substantially affected untilthe pH reached 5.3 (vertical line I), which suggest this may be thetolerance limit for propionate by A. acetophilum HS399.

As illustrated in Table 15, above, the model illustrated in the example3100 of FIG. 31 can be used to translate the extracellular pH andpropionic acid concentration to intracellular propionate concentrations.For example, this can be confirmed as a substantial equivalent to theconcentration obtained with the “initial propionate” approach (107.5 vs97.7 g/L). The results demonstrate that intracellular propionateconcentration may be a valid metric for propionic acid toxicity and usedas a metric that can be interpreted across different pH values andpropionate concentrations scenarios (see Table 15).

FIG. 32 is a 3D graphical representation of an example expression of thepropionic acid toxicity 3200. In this example, the propionic acidtoxicity 3200 is expressed against a grid that defines the thresholdtoxicity 3208 limit for different pH 3204 and extracellular propionatecombinations 3206. As an example, the graphing of the propionatetoxicity model can be a very useful tool for the design of the odd chainfatty acid production process. In this example, the propionic acidtoxicity 3200 represented as cytosolic propionate 3202 is controlled bythe extracellular pH 3204 and propionate concentration 3206. In oneimplementation, the 3D graph can be built through the integration ofHenderson and Hasselbalch equation. The grid intersection line 3208represents the lethal toxicity threshold validated experimentallyaccording to two different approaches.

In one implementation, a lower level of propionic toxicity may bedetermined, as described in the following example techniques:

Identify Lower Propionic Toxicity: Batch Vs. Fed-Batch

In one implementation, a 250 mL Erlenmeyer flask can be used tocultivate A. acetophilum HS399. In this example, sodium propionate caneither be batched or fed batch different propionate concentrations eachday. FIG. 33 is a graphical representation illustrating an example 3300where cell dry weight is measured each day to evaluate the impact of thepropionate feeding strategy in A. acetophilum HS399 growth. This exampleillustrates treatment of 3+3+3 (e.g., over respective days), where 3 g/Lpropionate is fed at 0, 24 and 48 hrs (9 g/L in total). This treatmentappears to provide better growth than a treatment fed 10 g/L propionateat 0 hr. These results illustrate that fed-batching may be a betterstrategy to lower propionic acid toxicity than batching all thepropionate at initial inoculation. Further, as illustrated in thisexample 3300, growth is decreased in a dose response manner with thedaily amount of propionate fed. In this example, the best growth isachieved with the lowest propionate dose (1 g/L d), but this treatmentstill shows growth inhibition when compared against the non-propionatecontrol treatment (0 g/L d).

Identify Lower Propionic Toxicity: Growth Vs. Lipid Phase

In one implementation, a 250 mL Erlenmeyer flask can be used tocultivate A. acetophilum HS399. In this example, 3 g/L of propionateacid can be fed during growth phase (3+0) or during lipid phase (0+3).Growth phase treatment is fed propionate at inoculation (0 hr), whilelipid phase treatment can be fed propionate upon depletion of theammonia from the media (˜24 hrs). Cell dry weights may be analyzed usinga filtration method. Ammonia can be measured using a Cedex bio-analyzer(Roche). Further, propionic acid can be measured using high performanceliquid chromatography. The fatty acid profile of the biomass can beanalyzed using gas chromatography.

In this implementation, total propionate consumed by the cell can becalculated based on the residual propionate on day 0 and subtracting thefinal residual propionate. The total propionate deposited in the biomasscan be calculated based on the final cell dry weight, multiplied by thetotal fatty acid (TFA) ratio in biomass, multiplied by the OCFA ratio inTFA, and multiplied by the molar factor of propionate in odd chain fattyacid, which averages at about 0.3. The propionate deposition can becalculated by dividing the propionate deposited by the propionateconsumed and expressed as percent.

FIG. 34 is a graphical representation illustrating results 3400 ofgrowth of A. acetophilum HS399 and residual ammonia when propionate wasfed in growth or lipid phases. As illustrated, the results 3400 identifythat propionic acid (0+3) is fed during the lipid phase, once theammonia was depleted from the media. As illustrated, this treatmentduring the lipid phase did not appear to provide a lag phase 3402 thatwas present when propionate was fed during the growth phase.Additionally, the propionic acid deposition is slightly improved whenpropionate is fed during the lipid phase than when propionate was fed inthe growth phase. These results 3400 illustrate that feeding propionateduring lipid phase may be preferred over feeding during the growth phasebecause of the lack of lag 3402 during the lipid phase, and an increasesof lipid deposition from 53 to 58.8%, as illustrated in Table 16 below.

TABLE 16 Propionic acid deposition into Aurantiochytrium acetophilumHS399 according to the stage in which propionate was fed. Propionate(g/L) Propionate Deposition (%) 0 (control)  0.0 ± 0.0 3 (growth) 53.0 ±1.0 0 + 3 (lipid) 58.8 ± 1.9

Example 9—Pathway Elucidation: A. acetophilum HS399 May not UsePropionic Acid as Sole Carbon Source

In one aspect, propionic acid may be either incorporated into A.acetophilum HS399 lipids or catabolized into the citric acid cycle. Inthis aspect, the propionate deposition may be controlled by both therate of lipid synthesis and the rate of propionate catabolism. Forexample, there are two main catabolic pathways that are responsible forpropionate oxidation into the citric acid cycle. The methyl-malonatepathway converts propionate into succinyl-CoA which enters the citricacid cycle. Alternatively, the methyl-citrate pathway, convertspropionate into succinate and pyruvate, both of which enter the citricacid cycle.

As identified by one or more the techniques described herein, themethyl-citrate pathway is anaplerotic because it releases twointermediates of the citric acid cycle. Because propionate is not ananaplerotic substrate, growth on propionate (e.g., as a sole carbonsource) may be sustained by an anaplerotic pathway such as themethyl-citrate. Non anaplerotic pathways, such as methyl-malonatepathway, which releases only one citric acid intermediate (succinyl-CoAintermediate), may not sustain growth on propionate as sole carbonsource.

In one implementation, the capacity of A. acetophilum HS399 to grow onpropionate as sole carbon can be determined to help identify thecatabolic pathway for propionate catabolism. In this implementation, anErlenmeyer flask (250 ml) can be inoculated (1% v/v) in triplicates at apH of 7. One treatment can be fed with non-lethal concentrations ofpropionate (e.g., 10 g/L), and other treatments can be fed glucose as apositive control, or no carbon substrate as a negative control. FIG. 35is a graphical representation of one implementation that illustratesresults 3500 of growth of A. acetophilum HS399 with different carbonsources. As illustrated, the results 3500 illustrate that the propionicacid treatment 3502 did not support growth above that of the starvedtreatment (negative control) 3504; while the glucose treatment 3506, asexpected, supported high growth.

Further, as illustrated in FIG. 36 , shows example results 3600 ofresidual propionate cultures of A. acetophilum HS399 fed differentcarbon sources. The results 3600 illustrate that the residual propionatedoes not decrease in response to cell uptake. These results 3500, 3600,suggest that the methyl-citrate cycle may not be active in A.acetophilum HS399. Therefore, propionic acid is most likely catabolizedvia a non anaplerotic methylmalonate pathway.

Example 10—Metabolic Intervention: Cyanocobalamin Regulating PropionateCatabolism in Microbials

As described above, methyl-malonate is likely the main propionatecatabolic pathway in A. acetophilum HS399. The enzyme methylmalonyl-CoAmutase converts a methylmalonyl-CoA into succinyl-CoA, in a reactionthat utilizes cyanocobalamin (vitamin B12) as a cofactor.

In one implementation, A. acetophilum HS399 can be sub-cultured in acyanocobalamin deprived media for at least 10-generations. FIG. 37 is agraphical representation of example results 3700 of sub-culturing A.acetophilum HS399 in a cyanocobalamin deprived media for over10-generations. The results 3700 illustrate that there is substantiallyno impact on growth of A. acetophilum HS399 between generations. In thisexample, the numerous generations help dilute the possibility ofpotential cell reserves of cyanocobalamin and demonstrate thatcyanocobalamin (Vitamin B12) is not likely essential. These results 3700also provide adequate evidence that A. acetophilum HS399 is not acyanocobalamin auxotroph.

In one implementation, the impact of methionine supplementation in thegrowth of A, acetophilum HS399 can be illustrated. In thisimplementation, methionine synthetase can use cyanocobalamin as acofactor to generate methionine from homocysteine. In thisimplementation, a growth media can be supplemented with 0 and 0.5 g/Lmethionine and the growth can be measured under the presence and absenceof cyanocobalamin (0.00037 μM). The methionine supplementation does notappear to have an impact in growth of A. acetophilum HS399 (FIG. 38 ),regardless of the presence or absence of cyanocobalamin. As an example,this may be consistent with A. acetophilum not being a cyanocobalaminauxotroph.

In one implementation, a cyanocobalamin deprived A. acetophilum HS399can be inoculated in triplicates (n=3) in the presence of propionate (3g/L), using three different concentrations of cyanocobalamin (0.37,0.00037, 0 μM). The propionic acid deposition by A. acetophilum HS399can be analyzed according to the method described above regardingPropionic Acid Deposition Rate. As illustrated in Table 17 below, a doseresponse increase in propionate deposition is identified, withdecreasing concentration of cyanocobalamin in the media. Of note, acyanocobalamin deprived media (0 μM) can result into 99.5% of thepropionate being incorporated into A. acetophilum HS399 lipids as OCFAs.For example, this may suggest that almost no propionate is oxidized,presumably due to the lack of methyl-malonyl-CoA mutase cofactor (e.g.,cyanocobalamin) blocking the pathway. The results shown in Table 17 areconsistent with the methyl citrate pathway not being active (asdescribed above), and methyl-malonate may be the primary catabolicpathway for propionic acid oxidation. A benefit associated withincreasing propionate deposition is decreasing the propionate used forthe production of OCFAs.

TABLE 17 Propionic acid deposition in response to cyanocobalamin(vitamin B12) concentration. Cyanocobalamin-Vit B₁₂ (μM) 0.37 0.00037 0Cell dry weight (g/L) 37.0 ± 1.4 32.0 ± 0.7 35.0 ± 1.2 OCFA (% DW)  2.7± 0.2 14.7 ± 0.2 25.0 ± 0.3 Initial propionate (g/L) 3   3   3   FinalPropionate (g/L)  0.06 0.1  0.36 OCFA (g/L)  1.0 ± 0.1  4.7 ± 0.1  8.7 ±0.4 Propionate (MW)/ 0.3 0.3 0.3 OCFA (MW) Propionate deposition 10.3 ±1.0 47.0 ± 0.9 99.9 ± 5.8 (% fed)

Example 11—Metabolic Intervention: Cyanocobalamin May not ImpactPropionic Acid Toxicity

In one implementation, in can be determined whether an increase ofpropionate deposition, through cyanocobalamin deficiency, has a negativeimpact in A. acetophilum HS399 growth and productivity. For example, atesting can show if propionic acid toxicity is affected by thecyanocobalamin concentration, because, as identified above,cyanocobalamin may not be essential for A. acetophilum HS399, at leastin the absence of propionate.

FIG. 38 is a graphical representation of one implementation illustratingexample results 3800 of cell dry weight and residual propionate, 3 g/Lpropionate. In this implementation, A. acetophilum HS399 can beinoculated at different concentrations of cyanocobalamin (0.00037, 0 μM)in triplicates (n=3). In this implementation, the pH can be maintainedat 7±1 in respective treatments. The initial 3 g/L propionate fed to thecultures is consumed within 72 hrs, during which insignificantdifferences are shown in growth due to the cyanocobalaminconcentrations, as illustrated by the results 3800. These resultssuggest that propionic acid catabolism may not be necessarily aprotection mechanism for propionic acid toxicity, for example, growthcan be inhibited as long as propionic acid is still present, as shown inthe lower half of the graph.

In one implementation, fermenters may be used to culture A. acetophilumHS399. In this implementation, propionic acid can be fed-batch in apH-auxostat mode (e.g., propionic acid used as pH titrant) 24 hrs afterinoculation. The fed batch system can appropriately control propionicacid toxicity by maintaining residual propionate concentration at2.0±0.1 g/L and the pH at 7±0.1. FIG. 39 is a graphical representationshowing results 3900 of the impact of cyanocobalamin in A. acetophilumHS399 growth and propionic acid consumption in 10 L Bioflo320fermenters. In this implementation, two treatments using differentcyanocobalamin concentrations (0.00185, 0 μM) are compared induplicates. As illustrated by the results 3900, the growth of bothtreatments is substantially equivalent, but the treatment withoutcyanocobalamin consumed 30% less propionic acid. As shown in Table 18below, the OCFA production may not be substantially affected by thecyanocobalamin concentration, for example, as long as propionic acid isfed on demand. However, as shown in Table 19 below, the propionatedeposition is elevated in the treatment without cyanocobalamin. In otherwords, as an example, cyanocobalamin deficiency may produce the sameOCFAs with a much smaller portion of the propionic acid. Further, forexample, Cyanocobalamin may not have any impact in propionic acidtoxicity and therefore it may be freely manipulated to improvedeposition rates.

TABLE 18 Impact of cyanocobalamin in the fatty acid profile ofAurantiochytrium acetophilum HS399 cultures grown in a 10 L Bioflo320fermenters. Cyanocobalamin (μM) — 0.37 0 Time (h) 0 73 73 Total FattyAcids (% DW) 26.7 54.7 ± 0.5 56.9 Fatty Acid Profile (% TFA) 13:0 0.0 1.4 ± 0.2 1.5 14:0 3.8  1.4 ± 0.0 1.3 15:0 1.1 42.6 ± 0.9 42.3 16:045.2 11.7 ± 1.6 10.8 17:0 0.4  8.6 ± 0.6 8.2 18:0 1.6  0.3 ± 0.0 0.322:5 (n-6) 7.7  3.6 ± 0.1 3.8 22:6 (n-3) 36.4 28.0 ± 1.0 29.1 Other FA3.8  2.5 ± 0.3 2.7 DHA (% DW) 9.7 15.3 ± 0.4 16.6 OCFA (% TFA) 1.5 52.6± 0.5 52.0

TABLE 19 Propionate deposition. Cyanocobalamin (μM)g_(propionate)/g_(biomass) Propionate deposition 0.37 0.18 ± 0.01 49.9%0 0.14 65.7%

Example 12—Mitigate Propionic Toxicity: Single Vs. Two-StageFermentation

In one implementation, the impact of propionate in two different growthmodes can be illustrated, using a two-stage and a single-stage growthmode, utilizing 10 L Bioflo fermenters, for example. In thisimplementation, a two-stage mode can comprise a first growth stage (0-24hrs), followed by a lipid phase (24-80 hrs), where nitrogen is notpresent. Thus, for example, in the two-stage mode substantially all ofthe nitrogen (5 g/L NH₃) can be fed during growth phase, which is thendepleted as it enters a lipid phase. Further, in this implementation,during the lipid phase, no additional nitrogen is fed, and the cellaccumulated the lipids. In the single stage mode, half of the nitrogen(2.5 g/L NH₃) can be batch fed, while the other half (2.5 g/L NH₃) canbe fed along with the glucose until the end of the fermentation. In thisimplementation, both treatments receive substantially the samenutrients; however, the single stage mode grows and accumulates lipidsin a coordinated way throughout the fermentation. The single stagesystem can be characterized by an early lipid accumulation.

Embodiments of the single stage system are described in detail inApplication No. PCT/US2018/29602 (Ganuza et al.), entitled SINGLE-STAGEFERMENTATION METHODS OF CULTURING MICROORGANISMS, filed on Apr. 26, 2018by the Applicant herein, which is incorporated herein in full byreference. The cultures can achieve higher lipid contents (˜32 hrs)sooner than the two-stage system (˜48 hrs) in the batch.

FIG. 40 is a graphical representation of two results 4000, 4050 of theimpact of propionic acid exposure to A. acetophilum HS399 growth and oddchain fatty acid production in 10 L Bioflo320 fermenters under single4000 or two-stage mode 4050. In this implementation, propionic acid canbe fed fed-batch in a pH-auxostat mode (e.g., propionic acid can also beused as the pH titrant) 24 hrs after inoculation to experimentaltreatments in order to illustrate the impact of the growth mode inpropionic acid toxicity. Each of the four treatments can be cultured induplicate (n=2) fermenters. As illustrated in Table 20 below, the growthrate can be determined, along with the amount of propionic acid fedthroughout the culture. As illustrated, propionic acid slows down thegrowth on both systems, but the two-stage system provides a bettergrowth rate than the single stage system in the presence of propionate.Additionally, the accumulation of OCFA in lipids (% TFA) is improveswhen propionic acid is fed in a two-stage process, for example, becauseno lipids (e.g., consisting of even chain fatty acids) may be producedin the absence of propionate (0-24 h). This implementation illustratesthat the traditional two stage approach to lipid accumulation may bepreferable to produce OCFAs.

TABLE 20 Impact of propionic acid exposure to Aurantiochytriumacetophilum HS399 biomass yield and productivity under single ortwo-stage mode. Single Stage Two Stage Propionic Propionic No Acid AcidNo Acid Acid Batch Time (h) 96 h 96 h 89 h 89 h Biomass Yield (g/L)148.8 82.5 163.6 125.8 Productivity (g/L/d) 37.4 20.7 44.3 34.1Double Ammonia-Propionic Acid/pH-Auxostat Process:

Based on the results from the implementations described above, in oneimplementation, a fermentation process can be used to produce OCFAsunder a two-stage growth mode, where ammonia is used as a nitrogensource and propionate is used as a promotor of OCFAs. Glucose can alsobe fed in a DO₂ stat mode in response to dissolved oxygen levels risingabove 15% saturation. Previously, as illustrated herein, fed-batch maybe preferred to batch because it can reduce propionic acid toxicity(e.g., “Mitigate propionic toxicity: Batch vs. fed-batch”). Further, asillustrated herein, propionic acid toxicity can be modulated with thepropionate concentration and the pH of the media (e.g., “Establishingpropionic acid toxicity limit”). Therefore, a pH-auxostat system can beused to maintain low residual concentrations of those nutrients in afed-batch mode, while controlling their toxicity through the pH setpoint.

Molar NH3/NaOH Ratio of the Fed

FIG. 41 is a graphical representation illustrating results 4100 of oneimplementation, where, much like the two-stage growth mode describedabove, ammonia can be fed merely during the growth phase. In thisimplementation, the results 4100 illustrate the impact of ammonia tosodium hydroxide ratio of the fed in the residual ammonia concentrationof a double auxostat culture of A. acetophilum HS399. In thisimplementation, ammonia can be gradually displaced from the media as thesodium hydroxide from the feed interferes with the titration of theammonia/pH-auxostat. The feeding of propionic acid can be postponeduntil the lipid phase (˜15-25 hrs) based on the results obtained inexample “Mitigate propionic toxicity: Growth vs. lipid phase,” describedabove.

As a result, in this implementation, a double ammoniapropionic/pH-auxostat can be used, where ammonia is fed on demand duringgrowth phase and propionate is fed on demand during the lipid phase(e.g., a two-stage process). To accommodate both auxostats, a transitioncan be used that would provide for the absence of ammonia during thelipid phase, which otherwise can interfere with the propionic acidauxostat titration. In this implementation, an inert base (e.g., sodiumhydroxide) can be blended with the ammonia feed in a specific ratio. Thesodium hydroxide titrates the pH irreversibly, gradually displacing theresidual ammonia in the medium until it is completely depleted, as shownby the results 4100. As illustrated, ammonia depletion can stop theauxostat feed, which indicates the end of the growth phase and thebeginning of the lipid phase. The rate of ammonia depletion, andtherefore the total ammonia fed, for example, can increase withdecreasing ammonia-sodium hydroxide ratio.

FIG. 42 is a graphical representation illustrating results 4200 of oneimplementation, applying the impact of ammonia to sodium hydroxide ratioof the fed in the total ammonia fed and biomass yields of a doubleauxostat culture of A. acetophilum HS399. In this implementation, theresults 4200 illustrate the total ammonia fed to the culture and theresulting biomass yields can also be controlled by the ammonia to sodiumhydroxide ratio. Further, the results 4200 indicate that the ammonia tosodium hydroxide ratio in the fed is a component for the control andoperation of the double auxostat system. For example, this control mayalso be applied with a different inert base, such us potassium hydroxideor calcium hydroxide.

Toxicity Control Through a pH Ramp

In one implementation, using the model described above in “Establishingpropionic acid toxicity limit” a pH to provide a desired balance of thetoxicity of both propionic acid and ammonia can be determined. Forexample, using this method, there may not be a specific pH that canaccommodate the toxicity of both nutrients. FIG. 43 is a graphicalrepresentation illustrating results 4300 of one implementation,describing the impact of pH set-point control in the transition ofammonia to propionic acid pH auxostat culture of A. acetophilum HS399.As illustrated, the pH ramp 4302 is used to sustain the growth of HS399.As illustrated, when A. acetophilum HS399 is grown in a doublepH-auxostat at a pH set-point of 7.0 4304, ammonia toxicity can inhibitits growth. Further, when A. acetophilum HS399 is grown at a pH of 5.54306 the ammonia toxicity can be greatly reduced, but the cells can beinhibited by propionate at the beginning of the lipid phase.Additionally, a ramp 4302 can be applied where pH set-point can besteadily increased from 5.5 to 7 between hour 5 to hour 17 offermentation, which can coincide with the end of the growth phase whenresidual ammonia was at a low point. In this example, the pH ramp canmitigate the toxicity of both nutrients and improve the growth rate forthe culture through the two-stage process 4308.

Activating and Maintaining the Propionic Acid pH Auxostat

FIG. 44 is a graphical representation illustrating results 4400 for A.acetophilum HS399 double pH-auxostat cultures, showing the impact ofsodium hydroxide supplementation into the glucose fed in the residualpropionic acid control in a pH-auxostat. In this implementation, thepropionic acid pH-auxostat can be implemented throughout the lipid phaseto improve the synthesis of OCFAs in A. acetophilum HS399. The lipidphase can begin when the ammonia feed naturally stops the addition. Atthis point, 1-3 g/L of residual propionate can be added into theculture. The propionate can be added as a salt (e.g., sodium propionate)or as an acid, in response to the simultaneous titration of sodiumhydroxide that is slowly (pH 7±0.2) introduced in the reactor. In thisimplementation, as soon as A. acetophilum HS399 starts consuming thepropionate, the pH raises, which provides for more propionate andactivation of the auxostat. In one implementation, the auxostat can bemaintained to keep the residual propionate levels constant, however, theresidual propionate may slowly decrease, which may be due to other cellmetabolites interfering with the titration, as shown in FIG. 44 . Inorder to overcome the slow decrease, 1-2 g of sodium hydroxide can beintroduced in respective liters of the glucose feed. In thisimplementation, the alkalization can help maintain the residualpropionate constant and avoid the early interruption of propionate fedthat may otherwise occur.

Example 13—Propionate Concentration and OCFA Titers

In one implementation, the variation of propionate fed toAurantiochytrium acetophilum HS399 cultures is reflected in the OCFAtiters of the final biomass. In this example, five treatments containingdifferent concentrations of propionate: 0, 2, 3, 4, 5 g/L respectively.Respective Erlenmeyer flasks (250 mL) are inoculated (1% v/v) intriplicates with a 24 h old culture of A. acetophilum HS399 andincubated in an orbital shaker at 180 rpm and 27° C.

Respective Erlenmeyer flasks contain 100 mL of a medium supplementedwith (g/L): dextrose (100), ammonium acetate (4.6), NaCl (12.5), MgSO₄7H₂O (2.5), KH₂PO₄ (0.5), KCl (0.5) and CaCl₂) (0.1). This medium alsocontains trace element solution (5 ml/L) and vitamin solution (1 ml/L).The trace element solution contains (g/L): EDTA di-sodium salt (6),FeCl₃ 6H₂O (0.29), H₂BO₃ (6.84), MnCl₂ 4H₂O (0.86), ZnCl₂ (0.06), NiSO₄6H₂O (0.052), CuSO₄ 5H₂O (0.002), Na₂MoO₄ H₂O (0.005). The vitaminsolution contains (mg/L): thiamine (100) and biotin (0.5).

In this example, respective culture materials are autoclaved (e.g., 121°C., 15 min) and the media is filter sterilized before use. A propionicacid stock solution (200 g/L) can be used as the fed propionic acid.Daily samples are collected to analyze the cell dry weight, residualglucose, culture pH and lipid and fatty acid composition of thecultures. Cell dry weights are analyzed by filtration (e.g., 0.2 μmfilter media) using a vacuum and washed with a solution of ammoniumbicarbonate. Residual glucose is analyzed using a colorimetric methodbased on glucose peroxidase activity. Biomass for lipid analysis iscentrifuged and washed using purified water. The washed biomass isfreeze dried. Total lipids are analyzed using Folch method (AOAC 996.06)and the FAMEs are analyzed by gas chromatography and flame ionizationdetection using nonadecanoic (C19:0) acid as an internal standard.

As shown in Table 21, the results illustrate that the differentpropionate concentrations yield different odd chain fatty acidconcentration. The results show that a wide range of OCFA concentrationin the oil can be produced by varying the propionate concentration inthe culture.

TABLE 21 Total lipids and fatty acid profile at time of harvest (96 h).Propionate (g/L) 0 2 3 4 5 Total Fatty Acids (% DW) 62.7 ± 0.8  54.8 ±5.6  56.2 ± 0.6  39.2 ± 12.4 41.0 ± 3.4  Fatty Acid Profile (% TFA) 13:00.0 ± 0.0 1.6 ± 0.1 2.6 ± 0.1 4.2 ± 0.4 5.1 ± 0.7 14:0 3.7 ± 0.0 3.0 ±0.2 2.4 ± 0.1 1.8 ± 0.3 1.9 ± 0.2 15:0 0.7 ± 0.0 25.2 ± 1.5  41.6 ± 0.9 51.6 ± 2.6  53.0 ± 0.4  16:0 51.5 ± 0.4  31.7 ± 1.3  17.0 ± 1.1  6.4 ±0.5 5.7 ± 0.5 17:0 0.3 ± 0.0 3.9 ± 0.4 6.7 ± 0.3 7.2 ± 0.3 6.6 ± 0.718:0 1.9 ± 0.3 0.9 ± 0.0 0.5 ± 0.0 0.1 ± 0.1 0.1 ± 0.1 22:5 (n-6) 7.4 ±0.1 5.1 ± 0.2 4.0 ± 0.2 2.7 ± 0.4 2.8 ± 0.1 22:6 (n-3) 33.0 ± 0.5  26.6± 0.6  23.4 ± 0.4  22.8 ± 1.9  21.9 ± 0.6  Other FA 1.4 ± 0.3 1.6 ± 0.01.7 ± 0.0 2.7 ± 1.2 2.2 ± 0.3 OCFA (% TFA) 0.9 ± 0.0 30.7 ± 1.8  50.8 ±1.1  63.0 ± 2.5  64.6 ± 0.3 

Example 14—Anaplerotic Process in 1000 L Pilot Fermenter

In one implementation, the processes described herein can be scaled upinto a larger pilot facility, such as a 1000 L pilot facility, induplicates fermenters (n=2). For example, the pilot facility may bereflective of the production in larger reactors of up to 180,000 L. Inthis implementation, the seed cultures can be scaled up from a 1 mLcryovial, into a 100 ml Flask, a 7 L wave bag, a 79 L fermenter, and thepilot fermenter with 800 L running volume. Thus, as an example, thecultures can be inoculated at 5% v/v, although 1% v/v inoculation mayalso be utilized. In this implementation, the fermenter cultures can beaerated at 1.3 vvm (volume of air per volume of culture per min) andagitated with Rushton impellers, for example, at 60 rpm in a dissolvedoxygen (DO₂) cascade control. As one example, rpm may increase, but notbe decreased, in response to DO₂, at levels below 10% saturation. Inthis implementation, the temperature can be controlled at 27±1° C.Further, the pressure can be controlled at (0.7-2 kg/cm²), for example,in response to excessive foaming or rpm at its high limit. Additionally,corn syrup 95DE can be fed in a D02-stat mode at 800 g/L glucose inresponse to DO₂ levels rising above 15%.

In this implementation, the glucose solution can be fed in a ramp from 0to 0.2 mL/min L initially (e.g., from 8 to 20 h elapsed fermentationtime), and from 20 h onwards the DO₂-stat triggered glucose feed canpulse 102 min at 0.3 mL/min L. The pH can be controlled with the ammoniafed at 5.5 during growth phase and propionic acid at 7.0 during lipidphase. The pH can be raised from 5.5 to 7.0 gradually between 5 and 17 hwhen residual ammonia is at a low point. Upon the ammonia running out ofthe culture (e.g., nitrogen feeding stopped), 1 g/L of NaOH can beslowly added while correcting the pH (7.0±0.5) with propionic acid(glacial). In this example, after this point, the pH can be controlledwith the propionic feed. As one example, foam can be controlled (e.g.,manually or automatically) using less than 1 mL/L of HODAG K-60defoamer.

In this implementation, the 1000 L pilot fermenter can be filled with400 L of batch media and fed with another 400 L of glucose feed media,and 40 L of ammonia fed, which may result in a final working volume of˜850 L. The example compositions of respective media are illustrated inin Table 22, and the trace metal solution is shown in Table 23, below,along with vitamin mix contained 100 mg of thiamin and 0.5 mg/L ofbiotin. The medium chemicals can be dissolved in the order listed on thetables, for example, and the pH of the media can be adjusted to 5.5using NaOH. The volume of the culture can be raised up to 345 L,accounting for another 35 L of condensation during a steam sterilizationprocess (121° C.×30 min). In this implementation, the batch mediaingredients can be filter sterilized, except for the vitamin mix, whichcan be filter sterilized into the reactor. Glucose fed can be pre-heatedthrough the jackets to mitigate excessive condensation during subsequentsteam sterilization (121° C.×30 min). The sodium hydroxide can be addedwithout sterilization once the sterilized tanks have cooled down to ˜40°C. The ammonia feed can also be prepared without sterilization.

TABLE 22 Medium formulation for production of OCFAs by Aurantiochytriumacetophilum HS399 in double pH-auxostat system. Batch NH3-FeedGlucose-Feed Chemicals Units media Media Media Ammonium sulfate g/L 3.8981.55 0 (NH₄)₂SO₄ Potassium phosphate g/L 5.0 0 0 KH₂PO₄ Magnesiumsulfate g/L 2.5 0 0 MgSO₄ 7•H₂O Potassium chloride KCl g/L 1.0 0 0Calcium chloride CaCl₂ g/L 0.2 0 0 Vitamin Mix mL/L 10 0 0 Trace MetalmL/L 50 0 0 Solution* ^(see table below) Corn syrup D95 g/L 37.5 0 1143Ammonium (29%) mL/L 0 238 0 NaOH g/L 0 64.2 2.0

TABLE 23 Formulation of the trace metals solution (TMS) RequiredQuantity Quantity Chemicals g/L (g for 1 L) EDTA disodium salt 6 6FeCl₃•6H₂O Iron (III) Chloride 0.29 0.29 hexahydrate (cloruro férrico)H₂BO₃ Boric acid 6.84 6.84 MnCl2•4H2O Manganese chloride tetrahydrate0.86 0.86 ZnCl₂ Zinc chloride 0.06 0.06 CoCl₂•6H₂O Cobaltous chloride0.026 0.026 NiSO₄•6H₂O Nickel (II) Sulfate Hexahydrate 0.052 0.052CuSO₄•5H₂O Copper (II) sulfate pentahydrate 0.002 0.002 Na₂MoO₄•2H₂OSodium molybdate dihydrate 0.005 0.005

In this implementation, the cultures can be monitored (e.g.,periodically or continuously) for T^(a), pH, DO2, oxygen uptake rate(OUR), rpm, and running volume. In this implementation, samples of celldry weight, lipids, fatty acids, residual glucose, ammonia andpropionate can be collected twice a day, or more.

In this implementation, the pilot (1000 L) fermenter may achieve 80 g/Lcell dry weight after 72 hrs of fermentation, as illustrated in theexample results 4500 of FIG. 45 . These results 4500 illustrate cell dryweight 4502 and residual glucose 4504 of A. acetophilum HS399 in a 1000L pilot fermenter. In this implementation, productivities of over 30 g/Lper day can be obtained, as illustrated in the results 4600 of FIG. 46 ,which illustrates the cumulative productivity of A. acetophilum HS399 ina 1000 L pilot fermenter.

Example 15

In some implementations, the performance of the reactor may be lowerthan its 10 L predecessor. This may be due to less than desired controlof the cultivation parameters, as illustrated by the results in FIGS.47A, 47B, 47C, 47D, which illustrate data resulting from substantiallycontinuous monitoring (a.k.a. online data) of A. acetophilum HS399 in a1,000 L pilot fermenter. Further, these results indicate that an oilcontaining 45% OCFAs could be industrially produced at scale usingAurantiochytrium sp. as shown in Table 24 in FIG. 48 using thetechniques described herein. That is, for example, data generated by oneor more sensors at the reactor or fermenter, such as pH sensor,dissolved oxygen sensor, off-gassing sensor, and others, can becontinuously monitored (e.g., or periodically monitored, andautomatically or manually recorded). In some implementations, the datamay be automatically recorded, and can be communicatively transmitted toa remote location, for example. In this way, the data can be collected,as illustrated in the examples of FIGS. 47A-D and 50 (below).

FIGS. 49A, 49B, 49C, 49D are graphical representations of exampleresults 4900 illustrating A. acetophilum HS399 growth in a 10 LBIOFLOW-320 fermenter using double pH-auxostat cultures for theproduction of odd chain fatty acids, growth productivity and lipidaccumulation (n=2). Further, FIG. 50 is a graphical representation ofexample results 5000 illustrating continuous monitoring of A.acetophilum HS399 double pH-auxostat cultures to produce odd chain fattyacids, DO2, glucose fed, pH, titrant addition and agitation (n=2).

Example 16—Anaplerotic Oils & Type 2 Diabetes

In one aspect, epidemiological data shows that odd chain fatty acids(OCFAs) in blood plasma inversely correlate with diabetes type 2(Forouhi et al. (2014): Lancet Diabetes Endocrinol, 2(10), 810-818;Santaren et al. (2014). Am. J. Clin. Nutr., 100(1), 1532-1540). In oneimplementation, A. acetophilum HS399 can be used to evaluate if OCFAshave an impact in glucose metabolisms.

FIG. 51 is a graphical representation of example results 5100illustrating growth and residual propionate in A. acetophilum HS399cultures that are subject to propionic anaplerosis triggered bycyanocobalamin. In FIG. 51 , the different letters (a, b) in respectivetime points indicate statistically significant differences according tothis implementation (p<0.05). FIG. 52 is a graphical representation ofexample results 5200 illustrating residual glucose in A. acetophilumHS399 cultures subject to propionic anaplerosis triggered bycyanocobalamin. In FIG. 52 , the different letters (e, f, g, h) in eachtime point indicate statistically significant differences according to at-student test (p<0.05).

In this implementation, a culture can be fed 3 and 9 g/L of odd numberedpropionic acid that is a product of the oxidation of longer chain fattyacids C15:0 and C17:0 in the presence or absence of cyanocobalamin (0 vs0.37 μM) in shake flask cultures. As described above, OCFAs anaplerosiscan merely take place in the presence of cyanocobalamin. In thisimplementation, the cell dry weight and residual propionate can bemonitored, and residual glucose in the media can be analyzed. As anexample, while the growth in the first 48 hrs may not be impacted by thecyanocobalamin, as illustrated by the results 5100 of FIG. 51 , theresidual glucose data indicates that glucose uptake rate may besignificantly (P<0.01 t-student) lower in the cyanocobalamin-anaplerotictreatment, as illustrated in the results 5200 of FIG. 52 . For example,these results indicate a link between OCFAs anaplerosis and glucosemetabolism, in support of a protective role of odd chain fatty acids(OCFAs) against diabetes type 2.

Example 17—OCFAs Promotors Alternative to Propionic Acid

In another aspect, alternative promoters for OCFA production may beutilized. Several implementations for use of alternative promoters aredescribed below and shown in Table 25 in FIG. 54 .

In one implementation, alternative promoters to propionic acid forproduction of OCFA may be implemented. According to techniques describedabove, A. acetophilum HS399 can accumulate OCFA in presence of propionicacid. Further, it may be beneficial to find alternative promotors toproduce OCFA that are less toxic to the model organisms or that aresimpler. In this implementation, pentanoate, heptanoate, yeast extract,proteose peptone, methionine, valine and isoleucine are evaluated fortheir capacity to induce the production of OCFAs. Erlenmeyer flasks canbe used and A. acetophilum HS399 can be cultured following the protocolsdescribed herein.

In this implementation, respective flasks can be supplemented withdifferent concentrations of the proposed promoters and the resultingbiomass harvested and analyzed for total lipid and fatty acid analyses.In this implementation, valine and isoleucine are identified as anitrogen and OCFAs source. Yeast extract, but not proteose peptone, isidentified as a precursor of OCFAs, presumably because proteose peptonehas a smaller proportion of the amino acids present in a free form thanyeast extract. As illustrated in FIG. 53 , it pentanoate and heptanoatemay be able to promote the production of OCFAs, but the toxicity ofheptanoic is higher than that of propionic acid (see result 5300).

Example 18—Anaplerotic Oils & Health Benefits

In one aspect, epidemiological data shows that odd chain fatty acids(OCFAs) in blood plasma inversely correlate with diabetes type 2(Forouhi et al. (2014): Lancet Diabetes Endocrinol, 2(10), 810-818;Santaren et al. (2014). Am. J. Clin. Nutr., 100(1), 1532-1540). In oneimplementation, A. acetophilum HS399 can be used to evaluate if OCFAshave an impact in glucose metabolisms.

In one aspect, because anaplerotic substrates can be used to restoreenergy balance in mitochondria, there is a wide range of pathologies towhich odd chain fatty acids have shown benefits. Namely, odd chain fattyacids have been experimentally used to treat the following conditions:

-   -   Genetic Metabolic disorders        -   Glut1 deficiency        -   Fatty acid oxidation disorder (FAOD)        -   Pyruvate carboxylase deficiency (Mochel et al., 2005)        -   Carnitine Palmitoyltransferase II deficiency (Roe et al.,            2008)        -   Rett syndrome (RTT)        -   Phenylketonuria (Roe & Mochel, 2006)        -   Adult Polyglucosan Body Disease (APBD) (Roe et al., 2010)        -   long-chain fat oxidation disorders (Roe et al., 2002)    -   Neurodegenerative diseases:        -   Epilepsy (Borges and Sonnewald, 2012)        -   Alzheimer's disease        -   Parkinson        -   Autism spectrum disorder (ASD)    -   Metabolic syndrome diseases        -   Diabetes type 2        -   Obesity        -   Cardiovascular disease

Additionally, there is some indication that odd chain fatty acids canhelp in building muscle and improving athlete metabolism. For example,vigorous physical effort might result in depletion of glucose andglycogen, in which case the main anaplerotic substrate comes from theprotein. Based on this process, one may hypothesize that odd chain fattyacids might spare the use of protein catabolism as anapleroticsubstrate. Obesity and fat burn conditions may also benefit from use ofodd chain fatty acids found in anaplerotic oils. For example, duringperiods of fat burn, odd chain fatty acid might restore the energyimbalance and help catalyze the energy generation from lipids. Forinstance, patients recovering from a surgical procedure might benefitfrom the OCFA anaplerosis.

Table 26, in FIG. 55 , illustrates various, commonly available naturalvegetable oils. Further, the table shows the concentrations of variousfatty acids available in the respective vegetable oils. As illustrated,the OCFA, particularly C15 and C17, are not found, or are found in traceamounts in natural vegetable oils.

Table 27 below compares the features of the two types of concentratedanaplerotic oils, synthetic anaplerotic oils (e.g., tripentanoin)against naturally produced anaplerotic oils from microorganisms.Synthetic oils such as triheptanoin and tripentanoin typically have highconcentrations of OCFAs. However, anaplerotic oil produced frommicroalgae, as described herein, possesses several advantages over thesynthetic triheptanoin and tripentanoin. As illustrated, the naturalanaplerotic oil produced by algae includes OCFA that are naturallypresent in our diet (C15:0 and C17:0), while triheptanoin syntheticallysynthesis OCFA of C5:0 and C7:0, which are not found in naturallyoccurring food sources.

Further, in this example, the anaplerotic oil produced by algae cancontain a substantial amount of DHA, which is a valuable nutraceutical.For example, DHA (docosahexaenoic acid) is a fatty acid that is commonlyfound in the meat of cold-water fish (e.g., tuna, salmon, cod, etc.).DHA has been found to improve early brain development in infants and mayimprove the vision and cognitive function development. Further, DHA hasbeen used for treating type 2 diabetes, coronary artery disease (CAD),dementia, depression, and attention deficit-hyperactivity disorder(ADHD), as well as improving vision and cognitive function in adults.Additionally, DHA can be converted into eicosapentaenoic acid (EPA) inthe body, which is used in the prevention and reversal of heart disease,stabilizing heart rhythm, asthma, cancer, painful menstrual periods, hayfever, lung diseases, systemic lupus erythematosus (SLE), and certainkidney diseases. Both EPA and DHA have been used in combination to treathigh cholesterol, high blood pressure, psoriasis, Raynaud's syndrome,rheumatoid arthritis, bipolar disorder, certain inflammations of thedigestive system (ulcerative colitis), and to prevent migraine headachesin teenagers.

TABLE 27 Comparison of synthetic anaplerotic oils with naturally-produced anaplerotic oils from microorganisms ANAPLEROTIC OILSTripentanoin Anaplerotic Dairy Fat Process Chemical SynthesisBiosynthesis Biosynthesis Type of OCFA Artificial Natural-DietaryNatural-Dietary C5:0; C 7:0 C15:0; C17:0 C15:0; C17:0 OCFA (% TFA) 10060 1.5 DHA (% TFA) 0 25 <1Synthetic Production of Odd Chain Fatty Acid and their Triacylglycerides

In some embodiments, odd chain fatty acids OCFAs and triacylglyceridescontaining OCFAs may be produced synthetically. As an example, thesynthesis of saturated C15 or C17 fatty acids can be accomplished usingdifferent chemical reactions/strategies, some of which are summarized inDiagram 1 (A-I). The example methods described below can be used forsynthesizing fatty acids esters as well by adopting appropriateprotection/de-protection strategies. The fatty acids or esters can thenbe used to synthesize triglycerides, the reaction can be catalyzed bybase or preferably enzymatically to obtain OCFA enriched triglycerides.The synthetic scheme for triglyceride synthesis is described in Diagrams1A-1I below.

A) Kumada Cross-Coupling

B) Negishi Cross-Coupling

C) Sonogashira Cross-Coupling

D) Stille Cross-Coupling

E) Suzuki Cross-Coupling

F) Alkyne Lithiation

G) Jones Oxidation

H) Arndt-Eistert Homologation

I) Triglyceride Synthesis (Chemical or Enzymatic Synthesis)

Diagrams 1A-1I Examples of Chemical Synthesis of Odd Chain Fatty Acidand their Cleavage in a Triacylglyceride.

In another embodiment, a representative synthesis of odd chain fattyacid triglycerides from commercially available starting materials isdescribed in Diagram 2, below. In this embodiment, octyl bromide isfirst converted into corresponding boronate ester, which is thensubjected to Suzuki coupling with 7-bromoheptanoic acid methyl ester toyield C15 methyl ester. The methyl esters are then converted totriglycerides by treating with glycerol. The fatty acids/esters can besynthesized using other methods described in Diagram 2 using othercommercially available starting materials.

Diagram 2 Example Reactions for the Synthetic Production of Odd ChainFatty Acid Triglyceride from Commercially Available Starting Materials.Human Use of Anaplerotic Oils

The widespread use of pentadecanoic (C15:0) and heptadecanoic acid(C17:0) levels in human blood plasma as biomarkers for low incidence ofdiabetes type 2 and cardiovascular disease challenges the prevailingdietary guidelines around saturated fatty acid intake. Recently, theprospective cohort study “PURE” concluded that dairy consumption,responsible for 45% of the total saturated fat intake, was associatedwith lower risks of cardiovascular disease and mortality, which echoesthe long debate around the French Paradox and the fact that dairy isalso the main dietary source of odd-chain fatty acids (OCFAs). Milk fathas C15:0 and C17:0 present only trace amounts (<2% total fatty acids,TFA) compared to the prevalent even-chain saturated fatty acids (˜70%TFA).

As described herein, natural oils that contains saturated fatty acidsthat are predominantly odd-chain (odd-chain fatty acids (OCFA)) may beused as a supplement to typical dietary intake of OCFA. In oneimplementation, as described herein, Aurantiochytrium acetophilum strainHS399 is capable of accumulating lipids of up to 85% of its weight, suchas when grown according to the techniques described herein (e.g., suchas heterotrophic conditions). A. acetophilum HS399 naturally producespalmitic acid (C16:0) (e.g., at 45% TFA) and docosahexaenoic acid (DHA,C22:6 n−3) (e.g., at 40% TFA). The techniques and systems describedherein describe a modified cultivation process (e.g., fermentation) thatcan produce C15:0 (e.g., up to or greater than 40% of TFA), C16:0 (e.g.,up to and greater than 15% of TFA), C17:0 (e.g., up to or greater than8% of TFA) and up to or greater than 28% DHA of TFA.

As an example, the techniques described herein may have the capacity toproduce, at industrial scale, a human nutritional oil that will helpisolate the effect of dietary even-chain versus dietary odd-chain fattyacids. For example, in some embodiments, the natural oil containing highconcentrations of OCFA, described herein, may produce OCFA of >49% TFA.Such a product can be used to expand use of anaplerotic oils as a foodadditive, and research into anaplerotic metabolism and biomarkers inepidemiology, and ultimately become an important nutritional supplement.

As described above, most OCFA is introduced into the human diet throughconsumption of dairy-related products. Dairy-related products also havephytanic acid, as do some ruminant animal fats, and certain fish.Phytanic acid in consumed foods can provide a type of marker for thesource of the OCFA when they are found together in a product, forexample, from dairy. In one implementation, a consumable may be producedthat comprises higher levels of OCFA, without the presence of Phytanicacid. That is, for example, the techniques described herein can producehigher levels of OCFA without producing phytanic acid. As such, in thisexample, the resulting biomass and/or oils can be used in food and willcomprise higher levels of OCFA without phytanic acid.

In one aspect, saturated fat with a higher concentration of OCFA alsohas a higher melting point. In one implementation, those saturated fatswith a melting point that is approximately 35 degrees Celsius or highercan contain a desired amount of OCFA, such as those that may be producedby the techniques described herein. In one implementation, thosesaturated fats with the elevated melting points (e.g., ≥35° C.) can beintroduced into human food. In one implementation, one or more portionof biomass resulting from techniques described herein, comprising thosesaturated fats with the elevated melting points (e.g., ≥35° C.), may beused for human consumption. In this way, for example, using saturatedfats with elevated melting points can result in consumption of elevatedamounts of OCFA. As one example, those saturated fats with OCFAconcentrations higher than ten percent will typically have a meltingtemperature greater than 35 degrees Celsius.

Example 19—Incorporating Anaplerotic Oil Rich in OCFA into Food

The average saturated fat (SFA) intake of a person based on a2,500-calorie diet is about 25-28 g SFA/person day (See for exampleWorld Health Organization; see also for example US Department of Health;Otto et al., 2012). It is typically recommended that saturated fatintake should be less than 10% of the total energy intake. Id. In oneaspect, consumption of about 26 g/person day of a microbial anapleroticoil (MAO), such as an algal anaplerotic oil (AAO) described above, cancontribute with approximately half of the average saturated fat intake(˜14 g SFA/person day). That is, for example, the MAO derived from oneor more techniques described herein may provide forty-five percent(e.g., or more) of the SFA per person day when consumed.

In one implementation, in this aspect, significant amounts of thedietary even chain saturate fat found in standard naturally occurringfoods (e.g., all-natural beef, as opposed to plant-based beefsubstitute) can be replaced with odd chain saturate fat from the MAO,for example, without significantly changing the dietary habits of theconsumer. For example, the inclusion levels of the MAO, comprisingapproximately 45% OCFAs, may depend on each product. However, generally,the dietary even chain saturated fat can be replaced with odd chainsaturated fat. As an example, if a whole milk serving (244 g) typicallyhas 4.6 g of saturate fat, a portion of the even-chain saturated fat canbe replaced with the MAO, as follows: 4.6 g of saturated fat inmilk/0.45 OCFA in the MAO=10.2 g of MAO can be used to replace theeven-chain fat with the OCFA. That is, for example, 10.2 g of the MAOreplaces the 4.6 g of saturated fat in the milk to have approximatelythe same amount of fatty acid present in the milk, except it would beOCFA instead of even-chain fatty acid.

In an implementation regarding standard naturally occurring foods, theMAO or AAO can be incorporated both as a direct food ingredient, as anutritional supplement, and used in many different food products as afood additive, such as those belonging to the categories listed below,and others not listed. As one example, any food that comprises fattyacid may be appropriate for replacement of the even-chain fatty acidwith the OCFA found in the MAO described herein. Further, the MAOdescribed herein may be incorporated into foods as an additive ornutritional supplement to provide benefits associated with consumptionof OCFA.

-   -   Dairy Products: Milk, cheese, cream, yogurt, butter, powder milk    -   Eggs and egg products    -   Spreads, sauces, dressings and condiments: mayonnaise,        margarine, cheese spreads, salad dressings, nut spreads    -   Meat products: Poultry, swine, cattle, lamb, lard, sausages,        cured meat, jerky,    -   Sports foods: energy bars, protein concentrates, protein        supplements    -   Snacks and crackers    -   Produce: fruits and vegetables    -   Drinks: sodas, juices, energy drinks etc.    -   Gelatins and puddings    -   Soups and broths    -   Frozen desserts    -   Seafood: fish, crustaceans, mollusks etc.    -   Plant based meat and seafood: tuna equivalent, burger equivalent        etc.    -   Pasta & Rice: spaghetti, noodles etc.    -   Bakery: brad, tortillas, pita, naan, cakes, desserts    -   Breakfast foods: cookies, corn flakes, pop corns, oats,        breakfast cereals, pastries and pie    -   Chocolates containing food    -   Culinary oils: cooking and dressing oil    -   Soy-based products: tofu, soy sauce, miso etc.    -   Soft and hard candy    -   Food additives: Olive oil conserve    -   Canned foods    -   Frozen foods    -   Infant formulae    -   Analogs to the products listed above

In another implementation regarding plant-based foods, one may identifythe amount of saturated OCFAs (e.g., tridecanoic, pentadecanoic, andheptadecanoic fatty acids) present in a standard food (e.g., ¼ pound ofnatural beef burger). One may then add an amount of MAO or AAO thatcontains the same amount of saturated OCFAs found in the standard foodto a target corresponding plant-based food (e.g., ¼ pound plant-basedburger). In this way, the plant-based substitute food (in this example,the plant-based burger) now contains the same amount of OCFAs that wouldbe contained in its standard food counterpart (in this example, thenatural beef burger) and thereby the plant-based substitute food becomesthe plant-based equivalent to its counterpart standard food. It shouldbe clearly understood that substantial benefit may also be derived fromadding an amount of MAO or AAO to the target plant-based substitute foodso that the amount of saturated OCFAs in the target plant-basedsubstitute food exceeds that contained in its standard food counterpart.It should also be clearly understood that substantial benefit may alsobe derived from adding an amount of MAO or AAO to the target plant-basedsubstitute food so that the amount of saturated OCFAs in the targetplant-based substitute food is lower than that contained it its standardfood counterpart.

In one implementation regarding plant-based foods, one may identify theamount of saturated fat present in a standard food (e.g., cow milk-basedbutter). One may then prepare a plant-based substitute food (e.g. soybased butter) and add an amount of MAO or AAO that contains an amount ofsaturated OCFAs to the plant-based food (e.g. soy based butter), whereinthe plant-based substitute food (in this example, the plant-basedbutter) now contains a lower amount of saturated OCFAs than thatcontained in its standard food counterpart (in this example, the cowmilk based butter). Thus, the plant-based butter substitute becomes ahealthier alternative to its counterpart standard butter.

In another implementation, one may add an amount of MAO or AAO to aplant-based food that does not naturally contain any OCFAs. For example,plant-based dairy products may contain zero OCFAs. One may then add anamount of MAO or AAO to the plant-based food (in this example, theplant-based dairy product) so that it now contains saturated OCFAs.

The invention disclosed herein may be applied in several ways. In oneembodiment, the saturated fat in a standard naturally occurring food maybe removed by processes such as separation with a solvent, separationvia filtration, molecular distillation, or any other means capable ofremoving saturated fat; then, the saturated OCFAs may be added to thetarget food product via the inclusion of the MAO or AAO. In anotherembodiment, the saturated fat in a standard naturally occurring food mayremain unchanged and then the saturated OCFAs may be added to the targetfood product via the inclusion of the MAO or AAO. And in anotherembodiment, one may determine a targeted daily amount of OCFAs toinclude in the target food and may then add that targeted daily amountof saturated OCFAs to the target food (this would apply when the targetfood initially contains zero OCFAs); or, alternatively, when the targetfood does contain some saturated OCFAs (but contains less OCFAs than thetargeted daily amount of OCFAs) one may add an amount of saturated OCFAsvia the inclusion of the MAO or AAO so that the total amount of OCFAs(naturally occurring OCFAs+OCFAs added via AAO/MAO) matches the targeteddaily amount of OCFAs.

A demonstration was performed to illustrate the addition of AlgalAnaplerotic Oil (AAO) to different groups of food suitable for animal(both human and non-human) consumption. Samples of food were weightedand then freeze dried for 48 h. Consequently, samples were weighted on adry basis and spiked with Algal Anaplerotic Oil as indicated in Tables28-30 below. Total lipids were analyzed using the Folch method (AOAC996.06) and the FAMES were analyzed by gas chromatography usingnonadecanoic (C19:0) acid as an internal standard. The AAO was added tosupply 20-30% of the highest inclusion levels for the AAO targetedamount of 6.0 g/day in a serving size of each of the food samples. InTables 28-30, the OCFA (% TFA) refers to the total amount of saturatedC13:0, C15:0 and C17:0 fatty acids in the particular food item.

Table 28 below shows an example of the fatty acid composition of varioustypes of milk: whole fat milk, low fat milk, almond milk, and soy milk.Specifically, as shown in the examples, OCFA comprised about 24% of theTFA in low fat milk; OCFA comprised about 26.3% TFA in almond milk; andOCFA comprised about 10% TFA in soy milk. In some embodiments, afteraddition of microbial anaplerotic oil to milk, the OCFA may compriseabout 1-30% of TFA of the milk. In other embodiments, the OCFA maycomprise about 2-5% of the TFA of the milk. In other embodiments, theOCFA may comprise about 5-10% of the TFA of the milk. In otherembodiments, the OCFA may comprise about 10-15% of the TFA of the milk.In other embodiments, the OCFA may comprise about 15-20% of the TFA ofthe milk. In other embodiments, the OCFA may comprise about 20-25% ofthe TFA of the milk. In other embodiments, the OCFA may comprise about25-30% of the TFA of the milk. It should be clearly understood thatsubstantial benefit may still be derived from higher or lower rates ofinclusion of OCFA in the milk.

In this example, it should be clearly understood that the milk productsmay be standard natural animal milk (from cows, goats, etc.) orplant-based substitute milk (e.g., soy or almond milk). Where the milkis a plant-based substitute milk, an amount of AAO or MAO may be addedin order to match or exceed the amount of saturated OCFAs present in thestandard naturally occurring milk counterpart.

TABLE 28 Fatty Acid Composition of Milk before and After AlgalAnaplerotic Oil Addition Low Fat Low Fat Whole Fat Milk Milk + AlmondAlmond + Soy Soy + Milk Control AAO Control AAO Control AAO TFA (% DW)24.1  6.0 10.7 9.7 18.5  22.6  27.3  C10:0 1.7 1.1 0.7 — — — — C12:0 2.52.1 1.3 — — — — C13:0 — — 0.6 — 0.7 — 0.2 C14:0 9.6 9.1 6.1 — 0.5 — 0.2C14:1 0.7 0.7 0.4 — — — — C15:0 1.0 1.0 18.7 — 20.7  0.1 7.8 C16:0 30.2 31.1  22.4 20.8  15.1  10.6  10.2  C16:1 1.6 1.7 1.2 1.7 1.1 — 0.1 C17:00.7 0.7 4.6 — 4.9 0.2 2.0 C17:1 — — — 0.4 0.2 — — C18:0 14.8  15.2  9.54.7 2.7 4.1 3.5 C18:1n9 31.1  31.0  19.5 — — 21.6  18.0  C18:2n6 4.5 4.22.8 72.3  40.2  54.1  45.1  C18:3n3 0.8 0.7 0.5 — — 8.4 7.0 C18:3n6 — —— — — — — C19:0 IS 6.9 25.8  14.3 15.4  7.9 7.1 6.2 C20:0 0.2 — — — —0.3 0.3 C20:1n9 0.6 1.4 — — — 0.2 0.2 C20:2 — — — — — — — C20:3n3 — — —— — — — C20:3n6 — — — — — — — C20:4n6 — — — — — — — C20:5n3 EPA — — — —0.3 0.3 0.4 C22:1n9 — — — — — — — C22:5n6 DPA — — 1.7 — 2.0 — 0.8C22:6n3 DHA — — 10.0 — 11.5  — 4.3 C24:1 & C22:5n3 — — — — — — — OCFA (%TFA) 1.7 1.7 24.0 — 26.3  0.3 10.0  Serving size (g) 240    240    240240    240    240    240    AAO per serving (g) — — 2.0 — 0.9 — 1.7 FatContent (% WW) 3.8 1.0 1.8 1.0 1.4 1.9 2.6

Table 29 below shows an example of the fatty acid composition of varyingtypes of dairy-containing products such as yogurt, ice cream, andchocolate. Specifically, as shown in the examples, OCFA comprised about28.2% of the TFA in yogurt; OCFA comprised about 13.1% of the TFA in icecream; and OCFA comprised about 5.8% of the TFA in chocolate. In someembodiments, after addition of microbial anaplerotic oil todairy-containing products, the OCFA may comprise about 5-30% of TFA ofthe dairy-containing product. In other embodiments, the OCFA maycomprise about 5-10% of the TFA of the dairy-containing product. Inother embodiments, the OCFA may comprise about 10-15% of the TFA of thedairy-containing product. In other embodiments, the OCFA may compriseabout 15-20% of the TFA of the dairy-containing product. In otherembodiments, the OCFA may comprise about 20-25% of the TFA of thedairy-containing product. In other embodiments, the OCFA may compriseabout 25-30% of the TFA of the dairy-containing product. It should beclearly understood that substantial benefit may still be derived fromhigher or lower rates of inclusion of OCFA in the dairy-containingproducts.

In this example, it should be clearly understood that thedairy-containing products may be standard natural dairy-containingproducts (i.e., containing milk from cows, goats, etc.) or plant-basedsubstitute dairy-containing products (i.e., containing soy milk oralmond milk). Where the dairy-containing product is a plant-basedsubstitute dairy-containing product, an amount of AAO or MAO may beadded in order to match or exceed the amount of saturated OCFAs presentin the standard naturally occurring dairy-containing counterpart.

It should be clearly understood that “dairy-containing products” mayalso include butter. For example, by starting with a plant-based milk(e.g., soy, almond, etc.) and adding an amount of MAO or AAO to it, aplant-based butter product may be created wherein the amount ofsaturated OCFAs in the plant-based butter substitute is lower thatcontained in its natural dairy-containing butter counterpart wherein thenatural dairy-containing butter counterpart was made with milk comingfrom an animal such as a cow or goat.

It should also be clearly understood that further substantial benefitmay be derived from adding MAO or AAO to non-dairy containing products,such as noodles, wherein the food contains vegetable oil, canola oil, orany other oil suitable for food production. As with the other examplescontained herein, an amount of MAO or AAO may be added to the non-dairycontaining product, such as noodles, in order to add a desired amount ofsaturated OCFAs thereto.

TABLE 29 Fatty Acid Composition of Dairy-Containing Products before andAfter Algal Anaplerotic Oil Addition Yogurt Yogurt + Ice Cream IceCream + Chocolate Chocolate + Control AAO Control AAO Control AAO TFA (%DW) 7.3 13.6 19.6  24.0  41.7  45.7  C10:0 1.2 1.0 1.8 1.4 — — C12:0 2.71.7 2.9 2.4 — — C13:0 — 0.7 0.1 0.4 — 0.1 C14:0 11.7  6.7 11.0  9.1 0.10.2 C14:1 0.8 0.5 0.8 0.6 — — C15:0 1.3 22.1 1.2 10.1  — 4.4 C16:0 35.8 22.5 35.5  30.0  26.7  24.9  C16:1 1.8 1.1 1.7 1.5 0.3 0.3 C17:0 0.8 5.40.7 2.6 0.2 1.2 C17:1 — 0.2 0.3 0.2 — — C18:0 14.0  7.5 14.1  11.3 35.6  32.2  C18:1n9 26.0  14.2 25.3  20.3  32.5  29.7  C18:2n6 3.2 1.83.8 3.1 3.1 2.8 C18:3n3 0.7 0.4 0.5 0.4 0.2 0.2 C18:3n6 — — — — — —C19:0 IS 22.4  11.5 8.2 6.5 3.8 2.7 C20:0 — — 0.2 0.1 1.1 1.0 C20:1n9 —— — — — — C20:2 — — — — — — C20:3n3 — — 0.2 0.2 — — C20:3n6 — — — 0.2 —— C20:4n6 — — — — — — C20:5n3 EPA — 0.4 — 0.2 0.2 0.2 C22:1n9 — — — — —— C22:5n6 DPA — 2.0 — 0.9 — 0.4 C22:6n3 DHA — 11.9 — 5.0 — 2.4 C24:1 &C22:5n3 — — — — — — OCFA (% TFA) 2.2 28.2 1.9 13.1  0.2 5.8 Serving size(g) 113    113 89   89   35   35   AAO per serving (g) — 1.5 — 1.9 — 2.1Fat Content (% WW) 2.6 3.9 9.9 12.0  38.4  44.5 

Table 30 below shows an example of the fatty acid composition of varyingtypes of animal products such as salmon meat, turkey meat, and imitationeggs (e.g., made of mung bean protein isolate or any other suitableplant-based material). Specifically, as shown in the examples, OCFAcomprised about 8.7% of the TFA in salmon meat; OCFA comprised about29.3% of the TFA in turkey meat; and OCFA comprised about 13.7% of theTFA in poultry eggs. In some embodiments, after addition of microbialanaplerotic oil to animal products, the OCFA may comprise about 8-30% ofTFA of the animal product. In other embodiments, the OCFA may compriseabout 8-10% of the TFA of the animal product. In other embodiments, theOCFA may comprise about 10-15% of the TFA of the animal product. Inother embodiments, the OCFA may comprise about 15-20% of the TFA of theanimal product. In other embodiments, the OCFA may comprise about 20-25%of the TFA of the animal product. In other embodiments, the OCFA maycomprise about 25-30% of the TFA of the animal product. It should beclearly understood that substantial benefit may still be derived fromhigher or lower rates of inclusion of OCFA in the animal products.

In this example, it should be clearly understood that the animalproducts or meat may be standard natural meat or plant-based substitutemeat. Where the meat is a plant-based substitute meat, an amount of AAOor MAO may be added in order to match or exceed the amount of saturatedOCFAs present in the standard naturally occurring meat counterpart. Itshould also be clearly understood that the word “meat” as used hereinrefers to any red meat, white meat, fish, and other seafood.

TABLE 30 Fatty Acid Composition of Animal Products before and AfterAlgal Anaplerotic Oil Addition Salmon Salmon + Turkey Turkey + EggsEggs + Control AAO Control AAO Control AAO TFA (% DW) 19.4  23.7 7.011.0  37.3  47.0  C10:0 — — — — — — C12:0 — 0.1 — — — — C13:0 — 0.2 —0.7 — 0.3 C14:0 1.8 1.7 0.7 0.8 — 0.3 C14:1 — — — — — — C15:0 0.2 6.8 —23.0  — 10.7  C16:0 11.4  10.9 17.4  12.9  5.2 5.8 C16:1 2.3 2.1 1.3 0.70.2 0.2 C17:0 0.2 1.7 0.4 5.6 0.1 2.6 C17:1 0.3 0.3 — — 0.2 0.1 C18:03.8 3.2 9.7 5.3 1.9 1.5 C18:1n9 41.8  35.8 27.6  13.4  61.3  47.4 C18:2n6 17.6  15.1 36.9  18.6  21.1  16.3  C18:3n3 5.0 4.3 2.9 1.6 7.45.7 C18:3n6 0.3 0.2 — — — — C19:0 IS 7.7 6.2 22.5  12.4  4.0 3.4 C20:00.3 0.3 — — 0.6 0.5 C20:1n9 2.9 2.5 — — 1.4 1.1 C20:2 1.4 1.2 — — — —C20:3n3 0.5 0.5 3.2 2.5 — — C20:3n6 0.6 0.5 — — — — C20:4n6 0.4 0.4 — —— — C20:5n3 EPA 3.1 2.8 — 0.4 0.4 0.4 C22:1n9 0.3 0.2 — — — — C22:5n6DPA — 0.7 — 2.3 — 1.0 C22:6n3 DHA 4.4 7.3 — 12.3  — 5.8 C24:1 & C22:5n31.5 1.3 — — 0.2 0.2 OCFA (% TFA) 0.4 8.7 0.4 29.3  0.1 13.7  Servingsize (g) 113    113 113    113    44   44   AAO per serving (g) — 1.5 —1.6 — 1.7 Fat Content (% WW) 5.2 6.5 1.3 2.7 11.0  14.9 Cyanocobalamin Affects Metabolism of Glucose and Propionate in Humansand Animals

Animal feed may be prepared to increase the cyanocobalamin concentrationtherein to ensure that the chicken can assimilate the propionate that isproduced from β-oxidation of odd chain fatty acids and therefore avoidpropionic acidemia. The metabolism of propionate requirescyanocobalamin, which is a cofactor of methyl-malonyl-CoA mutase thatproduces succinyl-CoA. Succinyl-CoA enters the citric acid cycle andcontributes to gluconeogenesis. The examples below also show howpropionate metabolism links to glucose metabolism as well as theimportance of cyanocobalamin to metabolize propionate.

Example 20

A demonstration was performed to illustrate the impact of cyanocobalaminon Aurantiochytrium acetophilum HS399 glucose metabolism.Aurantiochytrium acetophilum HS399 was cultivated in the presence andabsence of cyanocobalamin. In this demonstration, four treatments wereprepared with varying concentrations of propionate and cyanocobalamin (3g/L/0.00037 μM, 3 g/L/0 μM, 9 g/L/0.00037 μM and 9 g/L/0 μM). RespectiveErlenmeyer flasks (250 mL) were inoculated (1% v/v) in triplicates witha 24 h old culture of A. acetophilum HS399 and incubated in an orbitalshaker at 180 rpm and 27° C. The respective Erlenmeyer flasks contained100 mL of a medium supplemented with (g/L): dextrose (100), ammoniumacetate (4.6), NaCl (12.5), MgSO4 7H2O (2.5), KH2PO4 (0.5), KCl (0.5)and CaCl2 (0.1). This medium also contained trace element solution (5ml/L) and vitamin solution (1 ml/L). The trace element solutioncontained (g/L): EDTA di-sodium salt (6), FeCl3 6H2O (0.29), H2BO3(6.84), MnCl2 4H2O (0.86), ZnCl2 (0.06), NiSO4 6H2O (0.052), CuSO4 5H2O(0.002), Na2MoO4 H2O (0.005). The vitamin solution contained (mg/L):thiamine (100) and biotin (0.5). All culture materials were autoclaved(121° C., 15 min) and media was filter sterilized before use. Apropionate stock (200 g/L) was prepared using sodium propionate and wasused to supplement the cultures the respective propionate concentration.Daily samples were collected to analyze the cell dry weight, residualglucose, culture pH, lipid and fatty acid composition of the cultures.Cell dry weights were analyzed by vacuum filtration (0.2 μm) and washedwith a solution of ammonium bicarbonate. Residual glucose was analyzedusing a colorimetric method based on glucose peroxidase activity.Biomass for lipid analysis was centrifuged and washed using purifiedwater. The washed biomass was freeze dried. Total lipids were analyzedusing the Folch method (AOAC 996.06) and the FAMES were analyzed by gaschromatography using nonadecanoic (C19:0) acid as an internal standard.

FIG. 51 is a graphical representation of example results 5100illustrating growth and residual propionate in A. acetophilum HS399cultures that are subject to propionic anaplerosis triggered bycyanocobalamin. In FIG. 51 , the different letters (a, b) in respectivetime points indicate statistically significant differences according tothis implementation (p<0.05). FIG. 52 is a graphical representation ofexample results 5200 illustrating residual glucose in A. acetophilumHS399 cultures subject to propionic anaplerosis triggered bycyanocobalamin. In FIG. 52 , the different letters (e, f, g, h) in eachtime point indicate statistically significant differences according to at-student test (p<0.05).

In this implementation, a culture can be fed 3 and 9 g/L of odd numberedpropionic acid that is a product of the oxidation of longer chain fattyacids C15:0 and C17:0 in the presence or absence of cyanocobalamin (0 vs0.37 μM) in shake flask cultures. As described above, OCFAs anaplerosiscan merely take place in the presence of cyanocobalamin. In thisimplementation, the cell dry weight and residual propionate can bemonitored, and residual glucose in the media can be analyzed. As anexample, while the growth in the first 48 h may not be impacted by thecyanocobalamin, as illustrated by the results 5100 of FIG. 51 , theresidual glucose data indicates that glucose uptake rate may besignificantly (P<0.01 t-student) lower in the cyanocobalamin-anaplerotictreatment, as illustrated in the results 5200 of FIG. 52 . For example,these results indicate a link between OCFAs anaplerosis and glucosemetabolism. Cell dry weight reached the same titers independently of thecyanocobalamin concentration.

Example 21

A demonstration was performed to illustrate the impact of cyanocobalaminon Aurantiochytrium acetophilum HS399 propionate metabolism.Aurantiochytrium acetophilum HS399 was cultivated in the presence andabsence of cyanocobalamin. In this demonstration, two treatments wereprepared with varying concentrations of cyanocobalamin (370 nM and 0nM). Respective Erlenmeyer flasks (250 mL) were inoculated (1% v/v) inquintuplets with a 24 h old culture of A. acetophilum HS399 andincubated in an orbital shaker at 180 rpm and 27° C. The respectiveErlenmeyer flasks contained 100 mL of a medium supplemented with (g/L):dextrose (100), ammonium acetate (4.6), NaCl (12.5), MgSO4 7H2O (2.5),KH2PO4 (0.5), KCl (0.5) and CaCl2 (0.1). This medium also containedtrace element solution (5 ml/L) and vitamin solution (1 ml/L). The traceelement solution contained (g/L): EDTA di-sodium salt (6), FeCl3 6H2O(0.29), H2BO3 (6.84), MnCl2 4H2O (0.86), ZnCl2 (0.06), NiSO4 6H2O(0.052), CuSO4 5H2O (0.002), Na2MoO4 H2O (0.005). The vitamin solutioncontained (mg/L): thiamine (100) and biotin (0.5). All culture materialswere autoclaved (121° C., 15 min) and media was filter sterilized beforeuse. A propionate stock (200 g/L) was prepared using sodium propionateand was used to supplement the cultures with 16 g/L of propionate. Dailysamples were collected to analyze the cell dry weight, residual glucose,culture pH, lipid and fatty acid composition of the cultures. Cell dryweights were analyzed by vacuum filtration (0.2 μm) and washed with asolution of ammonium bicarbonate. Residual glucose was analyzed using acolorimetric method based on glucose peroxidase activity. Biomass forlipid analysis was centrifuged and washed using purified water. Thewashed biomass was freeze dried. Total lipids were analyzed using theFolch method (AOAC 996.06) and the FAMES were analyzed by gaschromatography using nonadecanoic (C19:0) acid as an internal standard.

FIGS. 56, 57 and 58 are a graphical representation of example resultsillustrating growth, residual glucose and propionate in A. acetophilumHS399 cultures that are subject to propionic anaplerosis triggered bycyanocobalamin. In FIG. 57 , the different letters (a, b) in respectivetime points indicate statistically significant differences according tothis implementation (p<0.01). These figures show that human andnon-human animals need to have a balanced diet which includes sufficientamounts of cyanocobalamin to ensure proper metabolism of propionate.

In this implementation, a culture can be fed 16 g/L of odd numberedpropionic acid that is a product of the oxidation of longer chain fattyacids C15:0 and C17:0 in the presence or absence of cyanocobalamin (0 vs370 nM) in shake flask cultures. As described above, OCFAs anaplerosiscan merely take place in the presence of cyanocobalamin. In thisimplementation, the cell dry weight and residual propionate can bemonitored, and residual glucose in the media can be analyzed. In thisimplementation, supplementation of a higher concentration ofcyanocobalamin did show a positive difference in cell dry weight. Theresults shown in FIG. 57 , the residual glucose data indicates thatglucose uptake rate may be significantly (P<0.01 t-student) lower in thecyanocobalamin-anaplerotic treatment. For example, these resultsindicate a link between OCFAs anaplerosis and glucose metabolism. FIG.58 shows that cultures deficient in cyanocobalamin couldn't oxidizepropionate through the citric acid cycle, causing a chronical higherresidual propionate in the culture.

While this disclosure describes anaplerotic oil production using themicroalgae A. acetophilum HS399, and the yeast-fungi Yarrowialipolytica, it should be appreciated that the method disclosed can beused with various other species of Aurantiochytrium, thraustochytrids,or other species of microorganisms including microalgae yeast, fungi andbacteria.

The term “microalgae” refers to microscopic single cell organisms suchas microalgae, cyanobacteria, algae, diatoms, dinoflagellates,freshwater organisms, marine organisms, or other similar single cellorganisms capable of growth in phototrophic, mixotrophic, orheterotrophic culture conditions. The term fungi refers to microscopicand macroscopic single cell organisms such us yeast and filamentousfungi.

In some embodiments, microalgae biomass, excreted products, or extractsmay be sourced from multiple types of microalgae, to make a compositionthat is beneficial when applied to plants or soil. Non-limiting examplesof microalgae that can be used in the compositions and methods of theclaimed subject matter comprise microalgae in the classes:Eustigmatophyceae, Chlorophyceae, Prasinophyceae, Haptophyceae,Cyanidiophyceae, Prymnesiophyceae, Porphyridiophyceae,Labyrinthulomycetes, Trebouxiophyceae, Bacillariophyceae, andCyanophyceae. The class Cyanidiophyceae includes species of Galdieria.The class Chlorophyceae includes species of Chlorella, Haematococcus,Scenedesmus, Chlamydomonas, and Micractinium. The class Prymnesiophyceaeincludes species of Isochrysis and Pavlova. The class Eustigmatophyceaeincludes species of Nannochloropsis. The class Porphyridiophyceaeincludes species of Porphyridium. The class Labyrinthulomycetes includesspecies of Schizochytrium and Aurantiochytrium. The class Prasinophyceaeincludes species of Tetraselmis. The class Trebouxiophyceae includesspecies of Chlorella. The class Bacillariophyceae includes species ofPhaeodactylum. The class Cyanophyceae includes species of Spirulina.

Non-limiting examples of microalgae genus and species that can be usedin the compositions and methods of the claimed subject matter include:Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphoracoffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var.punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var.tenuis, Amphora delicatissima, Amphora delicatissima var. capitata,Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus,Aurantiochytrium sp., Boekelovia hooglandii, Borodinella sp.,Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor,Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis,Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetocerossp., Chlamydomonas sp., Chlamydomas perigranulata, Chlorella anitrata,Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida,Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea,Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolate,Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var.actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri,Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var.aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata,Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna,Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorellapringsheimii, Chlorella protothecoides, Chlorella protothecoides var.acidicola, Chlorella regularis, Chlorella regularis var. minima,Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorellasaccharophila, Chlorella saccharophila var. ellipsoidea, Chlorellasalina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp.,Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii,Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgarisvar. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgarisvar. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorellavulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorellazofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcuminfusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp.,Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonassp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp.,Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliellagranulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva,Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliellaterricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliellatertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp.,Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp.,Galdieria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcuspluvialis, Hymenomonas sp., Isochrysis aff. galbana, Isochrysis galbana,Lepocinclis, Micractinium, Monoraphidium minutum, Monoraphidium sp.,Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Naviculaacceptata, Navicula biskanterae, Navicula pseudotenelloides, Naviculapelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp.,Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschiaclosterium, Nitzschia communis, Nitzschia dissipata, Nitzschiafrustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschiaintermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusillaelliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular,Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla,Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoriasubbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp.,Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp.,Pleurochrysis camerae, Pleurochrysis dentate, Pleurochrysis sp.,Porphyridium sp., Prototheca wickerhamii, Prototheca stagnora,Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii,Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcusopacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium,Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp.,Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmissp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiellafridericiana.

Taxonomic classification has been in flux for organisms in the genusSchizochytrium. Some organisms previously classified as Schizochytriumhave been reclassified as Aurantiochytrium, Thraustochytrium, orOblongichytrium. See Yokoyama et al. Taxonomic rearrangement of thegenus Schizochytrium sensu lato based on morphology, chemotaxonomiccharacteristics, and 18S rRNA gene phylogeny (Thrausochytriaceae,Labyrinthulomycetes): emendation for Schizochytrium and erection ofAurantiochytrium and Oblongichytrium gen. nov. Mycoscience (2007)48:199-211. Those of skill in the art will recognize thatSchizochytrium, Aurantiochytrium, Thraustochytrium, and Oblongichytriumappear closely related in many taxonomic classification trees formicroalgae, and strains and species may be re-classified from time totime. Thus, for references throughout the instant specification forSchizochytrium, it is recognized that microalgae strains in relatedtaxonomic classifications with similar characteristics toSchizochytrium, such as Aurantiochytrium, would reasonably be expectedto produce similar results.

In some embodiments, the microalgae may be cultured in phototrophic,mixotrophic, or heterotrophic culture conditions in an aqueous culturemedium. The organic carbon sources suitable for growing microalgaemixotrophically or heterotrophically may comprise: acetate, acetic acid,ammonium linoleate, arabinose, arginine, aspartic acid, butyric acid,cellulose, citric acid, ethanol, fructose, fatty acids, galactose,glucose, glycerol, glycine, lactic acid, lactose, maleic acid, maltose,mannose, methanol, molasses, peptone, plant based hydrolysate, proline,propionic acid, ribose, saccharose, partial or complete hydrolysates ofstarch, sucrose, tartaric, TCA-cycle organic acids, thin stillage, urea,industrial waste solutions, yeast extract, and combinations thereof. Theorganic carbon source may comprise any single source, combination ofsources, and dilutions of single sources or combinations of sources. Insome embodiments, the microalgae may be cultured in axenic conditions.In some embodiments, the microalgae may be cultured in non-axenicconditions.

In one non-limiting embodiment, the microalgae of the culture in anaqueous culture medium may comprise Chlorella sp. cultured inmixotrophic conditions comprising a culture medium primary comprised ofwater with trace nutrients (e.g., nitrates, phosphates, vitamins, metalsfound in BG-11 recipe [available from UTEX The Culture Collection ofAlgae at the University of Texas at Austin, Austin, Tex.]), light as anenergy source for photosynthesis, and organic carbon (e.g., acetate,acetic acid) as both an energy source and a source of carbon. In someembodiments, the culture media may comprise BG-11 media or a mediaderived from BG-11 culture media (e.g., in which additional component(s)are added to the media and/or one or more elements of the media isincreased by 5%, 10%, 15%, 20%, 25%, 33%, 50%, or more over unmodifiedBG-11 media). In some embodiments, the Chlorella may be cultured innon-axenic mixotrophic conditions in the presence of contaminatingorganisms, such as but not limited to bacteria. Additional detail onmethods of culturing such microalgae in non-axenic mixotrophicconditions may be found in WO2014/074769A2 (Ganuza et al.), herebyincorporated by reference.

In some embodiments, by artificially controlling aspects of themicroalgae culturing process such as the organic carbon feed (e.g.,acetic acid, acetate), oxygen levels, pH, and light, the culturingprocess differs from the culturing process that microalgae experiencesin nature. In addition to controlling various aspects of the culturingprocess, intervention by human operators or automated systems occursduring the non-axenic mixotrophic culturing of microalgae throughcontamination control methods to prevent the microalgae from beingoverrun and outcompeted by contaminating organisms (e.g., fungi,bacteria). Contamination control methods for microalgae cultures areknown in the art and such suitable contamination control methods fornon-axenic mixotrophic microalgae cultures are disclosed inWO2014/074769A2 (Ganuza et al.), hereby incorporated by reference. Byintervening in the microalgae culturing process, the impact of thecontaminating microorganisms can be mitigated by suppressing theproliferation of containing organism populations and the effect on themicroalgal cells (e.g., lysing, infection, death, clumping). Thus,through artificial control of aspects of the culturing process andintervening in the culturing process with contamination control methods,the microalgae culture produced as a whole and used in the describedinventive compositions differs from the culture that results from amicroalgae culturing process that occurs in nature.

In some embodiments, during the culturing process the microalgae culturemay also comprise cell debris and compounds excreted from the microalgaecells into the culture medium. The output of the microalgae culturingprocess provides the active ingredient for composition that is appliedto plants for improving yield and quality without separate addition toor supplementation of the composition with other active ingredients notfound in the mixotrophic microalgae whole cells and accompanying culturemedium from the culturing process such as, but not limited to:microalgae extracts, macroalgae, macroalgae extracts, liquidfertilizers, granular fertilizers, mineral complexes (e.g., calcium,sodium, zinc, manganese, cobalt, silicon), fungi, bacteria, nematodes,protozoa, digestate solids, chemicals (e.g., ethanolamine, borax, boricacid), humic acid, nitrogen and nitrogen derivatives, phosphorus rock,pesticides, herbicides, insecticides, enzymes, plant fiber (e.g.,coconut fiber).

While OCFA production increase has been described herein in terms ofalgae, fungus yeast, and other microbial, the techniques and systemsdescribed herein may not be limited merely to these types of organisms.In one aspect, there are some plant sources that naturally contain OCFA,specifically C15 and C17, which could therefore be genetically modifiedto increase production of these OCFAs. That is, for example, much likewith the production of OCFAs in algae, fungus and yeast, the plantgenetic code may be modified to increase the already naturally occurringOCFA. As an example, the genetic code that increases OCFA production inmicroorganisms may be identified, and, using gene splicing techniques,such as CRISPR-type techniques, the OCFA code may be inserted intocertain plants. Additionally, genetic trait selection may be undertakento identify and breed plants that express improved OCFA production,resulting in a plant that has higher OCFA production. Also, acombination of genetic code manipulation and trait selection may be usedto produce plants with improved OCFA production.

In addition to microalgae, some plants are able to serve as additionalnatural sources of long odd chain fatty acids. As shown in FIG. 55 , asTable 26, there are several plants that naturally contain C15:0 fattyacid, including, but not limited to: grape, silybum marianum (athistle), wheat germ, and rapeseed. Additionally, several plantsnaturally contain C17:0 fatty acid, including, but not limited to:safflower, grape, silybum marianum, hemp, sunflower, wheatgerm, pumpkinseed, almond, rapeseed, and peanut. Typically, these plants merelyproduce trace levels of C15:0 and C17:0.

Higher plants do not typically produce odd chain fatty acids (OCFAs) atcommercially significant levels, however several plants (e.g., seeplants in Table 16) present a metabolic pathway capable of synthetizingOCFAs. For example, the alpha oxidation pathway is a catabolic routetypically associated with the degradation of β-methyl branched fattyacids (see ref 5-Buchhaupt et al., 2014). The enzyme α-oxygenase (αDOX),a heme protein, introduces an oxygen molecule to the α-C of a fattyacid, leading to a decarboxylation. An aldehyde dehydrogenase enzymecompletes the oxidation of the resulting aldehyde to the correspondingfatty acid with one less carbon atom. This pathway eliminates the methylgroup of the branched fatty acids, resulting in a straight chain (even)fatty acid, but it could also eliminate the methyl group of an evenchain fatty acid, which would result in the production of OCFAs (seeref. 8-Takahashi et al., 1992). This pathway has been described in peaplants (see ref. 7-Shine and Stumpf, 1974), tobacco leaves, cucumber,potato (see ref 6-Hamberg et al., 1999), and could potentially beoverexpressed in other plants, including oleaginous crops (e.g., soy,canola, flax, sunflower etc.), and directed to the production of OCFAsin plants. OCFAs production in oleaginous crops would benefit from theproductivities and infrastructure available for such agriculturalcommodities.

All references, including publications, patent applications, andpatents, cited herein, are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Allprovided ranges of values are intended to include the end points of theranges, as well as values between the end points.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents.

The inventive concepts described herein include all modifications andequivalents of the subject matter recited in the claims and/or aspectsappended hereto as permitted by applicable law.

REFERENCES

-   1. Weitkunat, K., Schumann, S., Nickel, D., Hornemann, S.,    Petzke, K. J., Schulze, M. B., . . . Klaus, S. (2017). Odd-chain    fatty acids as a biomarker for dietary fiber intake: a novel pathway    for endogenous production from propionate. The American Journal of    Clinical Nutrition, 105(6), ajcn 152702.    https://doi.org/10.3945/ajcn.117.152702.-   2. Řezanka, T., & Sigler, K. (2009). Odd-numbered very-long-chain    fatty acids from the microbial, animal and plant kingdoms. Progress    in Lipid Research, 48(3-4), 206-238.    https://doi.org/10.1016/j.plipres.2009.03.003.-   3. Chaung, K.-C., Chu, C.-Y., Su, Y.-M., & Chen, Y.-M. (2012).    Effect of culture conditions on growth, lipid content, and fatty    acid composition of Aurantiochytrium mangrovei strain BL10. AMB    Express, 2(1), 42. https://doi.org/10.1186/2191-0855-2-42.-   4. Fan K W, Chen F, Jones E B, Vrijmoed L L. Eicosapentaenoic and    docosahexaenoic acids production by and okara-utilizing potential of    thraustochytrids. J Ind Microbiol Biotechnol. 2001; 27    (June):199-202. doi:10.1038/sj.jim.7000169.-   5. Zhu, L., Zhang, X., Ji, L., Song, X., & Kuang, C. (2007). Changes    of lipid content and fatty acid composition of Schizochytrium    limacinum in response to different temperatures and salinities.    Process Biochemistry, 42(2), 210-214.    https://doi.org/10.1016/j.procbio.2006.08.002.-   6. Buchhaupt, M., Kahne, F., Etschmann, M. M. W. & Schrader, J.    Chapter 37—Biotechnological Production of Fatty Aldehydes. Flavour    Science (Elsevier Inc., 2014).    doi:10.1016/B978-0-12-398549-1.00037-4.-   7. Hamberg, M., Sanz, a & Castresana, C. α-Oxidation of fatty acids    in higher plants. J. Biol. Chem. 274, 24503 (1999).-   8. Shine, W. E. & Stumpf, P. K. Fat Metabolism in Higher Plants    Recent Studies on Plant a-Oxidation Systems. Arch. Biochem. Biophys.    147-157 (1974).-   9. Takahashi, T., Takahashi, H., Takeda, H. & Shichiri, M.    Alpha-oxidation of fatty acids in fasted or diabetic rats. Diabetes    Res. Clin. Pract. 16, 103-108 (1992).-   10. Park Y K, Dulermo T, Amaro R L, Nicaud J M. Optimization of odd    chain fatty acid production by Yarrowia lipolytica. Biotechnol    Biofuels. 2018; 11(158):1-12. doi: 10.1186/s13068-018-1154-4.

Although a particular feature of the disclosed techniques and systemsmay have been disclosed with respect to only one of severalimplementations, such feature may be combined with one or more otherfeatures of the other implementations as may be desired and advantageousfor any given or particular application. Also, to the extent that theterms “including”, “includes”, “having”, “has”, “with”, or variantsthereof are used in the detailed description and/or in the claims, suchterms are intended to be inclusive in a manner similar to the term“comprising.”

This written description uses examples to disclose the claimed subjectmatter, including the best mode, and also to enable one of ordinaryskill in the art to practice the claimed subject matter, includingmaking and using any devices or systems and performing any incorporatedmethods. The patentable scope of the inventive concepts, describedherein, are defined by the claims, and may include other examples thatoccur to those skilled in the art. Such other examples are intended tobe within the scope of the claims if they have structural elements thatare not different from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal language of the claims.

In the specification and claims, reference will be made to “animals.” Asused herein, “animals” refers to members of the kingdom Animalia, whichincludes humans and non-human animals (e.g., poultry, fish, etc.).

In the specification and claims, reference will be made to “food.” Asused herein, “food” refers to consumable products, in solid form (e.g.,fish meat, poultry meat, yogurt, eggs, etc.), liquid (e.g., milk,juice), or a mixture of solid and liquid forms (e.g., ice cream). Theword “food” also refers generically to either standard naturallyoccurring foods or plant-based substitute foods.

In the specification and claims, reference will be made to a number ofterms that have the following meanings. The singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. Approximating language, as used herein throughout thespecification and claims, may be applied to modify a quantitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” is not to be limited to the precisevalue specified. In some instances, the approximating language maycorrespond to the precision of an instrument for measuring the value.Moreover, unless specifically stated otherwise, a use of the terms“first,” “second,” etc., do not denote an order or importance, butrather the terms “first,” “second,” etc., are used to distinguish oneelement from another.

As used herein, the terms “may” and “may be” indicate a possibility ofan occurrence within a set of circumstances; a possession of a specifiedproperty, characteristic or function; and/or qualify another verb byexpressing one or more of an ability, capability, or possibilityassociated with the qualified verb. Accordingly, usage of “may” and “maybe” indicates that a modified term is apparently appropriate, capable,or suitable for an indicated capacity, function, or usage, while takinginto account that in some circumstances the modified term may sometimesnot be appropriate, capable, or suitable. For example, in somecircumstances an event or capacity can be expected, while in othercircumstances the event or capacity cannot occur—this distinction iscaptured by the terms “may” and “may be.”

The best mode for carrying out the claimed subject matter has beendescribed for purposes of illustrating the best mode known to theapplicant at the time and enable one of ordinary skill in the art topractice the claimed subject matter, including making and using devicesor systems and performing incorporated methods. The examples areillustrative only and not meant to limit the claimed subject matter, asmeasured by the scope and merit of the claims. The claimed subjectmatter has been described with reference to preferred and alternateembodiments. Obviously, modifications and alterations will occur toothers upon the reading and understanding of the specification. It isintended to include all such modifications and alterations insofar asthey come within the scope of the appended claims or the equivalentsthereof. The patentable scope of the inventive concepts, describedherein, are defined by the claims, and may include other examples thatoccur to one of ordinary skill in the art. Such other examples areintended to be within the scope of the claims if they have structuralelements that do not differentiate from the literal language of theclaims, or if they include equivalent structural elements withinsubstantial differences from the literal language of the claims.

What is claimed is:
 1. A food additive comprising anaplerotic oilderived from a microorganism cultured aerobically with propionate or aprecursor thereof, wherein the anaplerotic oil comprises saturatedtridecanoic (C13:0), pentadecanoic (C15:0), and heptadecanoic (C17:0)odd-chain fatty acids (OCFA), wherein: at least 5% of the anapleroticoil from the microorganism's total fatty acid (TFA) content is saturatedtridecanoic (C13:0), pentadecanoic (C15:0), and heptadecanoic (C17:0)OCFA; and at least 2.2% cell dry weight (CDW) of the biomass from themicroorganism is saturated tridecanoic (C13:0), pentadecanoic (C15:0),and heptadecanoic (C17:0) OCFA.
 2. The food additive of claim 1, whereinthe microorganism is cultured aerobically with propionate.
 3. The foodadditive of claim 2, wherein pentadecanoic acid makes up greater than18% of the TFA content of the anaplerotic oil and/or heptadecanoic acidmakes up greater than 5% of the TFA content of the anaplerotic oil. 4.The food additive of claim 2, wherein saturated tridecanoic (C13:0),pentadecanoic (C15:0), and heptadecanoic (C17:0) OCFA comprise a rangeof 5% to 60% of the TFA content of the anaplerotic oil.
 5. The foodadditive of claim 2, wherein the microorganism is a species from theclass Labyrinthulomycetes.
 6. The food additive of claim 5, whereinmicroorganism is a thraustochytrid.
 7. A food for consumption by humanand non-human animals, said food comprising anaplerotic oil derived froma microorganism cultured aerobically with propionate or a precursorthereof, wherein the anaplerotic oil comprises saturated tridecanoic(C13:0), pentadecanoic (C15:0), and heptadecanoic (C17:0) odd-chainfatty acids (OCFA).
 8. The food of claim 7, wherein the microorganism iscultured aerobically with propionate.
 9. The food of claim 8, wherein:the food is a dairy product; and the % total fatty acid (% TFA) ofsaturated tridecanoic (C13:0), pentadecanoic (C15:0), and heptadecanoic(C17:0) OCFA is at least 5.5% greater than the % TFA of saturatedtridecanoic (C13:0), pentadecanoic (C15:0), and heptadecanoic (C17:0)OCFA in a substantially identical dairy product that does not containthe anaplerotic oil.
 10. The food of claim 8, wherein the microorganismis a species from the class Labyrinthulomycetes.
 11. The food of claim10, wherein microorganism is a thraustochytrid.
 12. A processed foodproduct or food component comprising a food additive, the food additivecomprising anaplerotic oil derived from a microorganism culturedaerobically with propionate or a precursor thereof, wherein theanaplerotic oil comprises saturated tridecanoic (C13:0), pentadecanoic(C15:0), and heptadecanoic (C17:0) odd-chain fatty acids (OCFA).
 13. Theprocessed food product or food component of claim 12, wherein themicroorganism is cultured aerobically with added propionate.
 14. Theprocessed food product or food component of claim 13, wherein: theprocessed food product or food component is a dairy product; and the %total fatty acid (% TFA) of saturated tridecanoic (C13:0), pentadecanoic(C15:0), and heptadecanoic (C17:0) OCFA is at least 5.5% greater thanthe % TFA of saturated tridecanoic (C13:0), pentadecanoic (C15:0), andheptadecanoic (C17:0) OCFA in a substantially identical dairy productthat does not contain the anaplerotic oil.
 15. The processed foodproduct or food component of claim 13, wherein the microorganism is aspecies from the class Labyrinthulomycetes.
 16. The processed foodproduct or food component of claim 15, wherein microorganism is athraustochytrid.
 17. The food additive of claim 1, wherein: at least2.7% CDW of the biomass from the microorganism is saturated tridecanoic(C13:0), pentadecanoic (C15:0), and heptadecanoic (C17:0) OCFA.
 18. Thefood additive of claim 1, wherein: at least 14.7% CDW of the biomassfrom the microorganism is saturated tridecanoic (C13:0), pentadecanoic(C15:0), and heptadecanoic (C17:0) OCFA.
 19. The food additive of claim1, wherein: at least 25% CDW of the biomass from the microorganism issaturated tridecanoic (C13:0), pentadecanoic (C15:0), and heptadecanoic(C17:0) OCFA.
 20. The food additive of claim 1, wherein: up to 39.25%CDW of the biomass from the microorganism is saturated tridecanoic(C13:0), pentadecanoic (C15:0), and heptadecanoic (C17:0) OCFA.